pharmacoscan assay 96-array format automated...

PharmacoScan™ Assay 96-Array Format Automated Protocol USER GUIDE

for Beckman® Biomek FXP (Windows® 7)

Catalog Numbers 913025

Publication Number 703472

Revision 1

For Research Use Only. Not for use in diagnostic procedures.

The information in this guide is subject to change without notice.

DISCLAIMER

TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Important Licensing Information

This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses.

Corporate entity

Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288

TRADEMARKS

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Jitterbug is a trademark of Boekel Scientific. Microsoft, Excel, and Windows are either registered trademarks or trademarks of Microsoft Corporation in the United States and/or other countries. QIAGEN, CoralLoad, and REPLI-g are registered or registration-pending trademarks of the QIAGEN Group. Beckman Coulter, and Biomek are either registered trademarks or trademarks of Beckman Coulter, Inc. TRobot is a trademark of Biometra GmbH. DNA Engine Tetrad, Bio-Rad, Microseal, and Hard-Shell are registered trademarks of Bio-Rad Laboratories, Inc.

©2017 Thermo Fisher Scientific Inc. All rights reserved.

PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP (Windows® 7) Use

Contents

CHAPTER 1 The PharmacoScan™ Assay 96-Array Format Automated Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP . . 11

Running multiple plate workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

CHAPTER 2 Genomic DNA preparation and requirements. . . . . . . . . 14

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Sources of genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

General requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Special requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Assessing the quality of genomic DNA using 1% Agarose E-gels . . . . . . . . . . . . . . . . . . 15

Genomic DNA extraction/purification methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Genomic DNA cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Genomic DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Equipment, consumables and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

1. Thaw samples and control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2. Quantitate and dilute test sample gDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

3. Aliquot the diluted samples and the controls DNA 1 and DNA 2 . . . . . . . . . . . . . . . . . 19

4. Freeze or proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5. Create a Batch Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

CHAPTER 3 Preparation before you start . . . . . . . . . . . . . . . . . . . . . . 22

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

PharmacoScan™ Assay arrays, reagents, and GeneTitan consumables required . . . . . . . . 22

Reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Requirements and recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Room temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Special requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Thermal cycler recommendations and protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

r Guide 3

Contents

PCR plate type by thermal cycler for mPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Oven recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Plate centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Plate shakers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Equipment care and calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Seal, vortex, and spin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

About the reagents and master mix preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Pipettes and pipetting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Matrix™ 25 mL reagent reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Equipment, consumables, and reagents required for mPCR preparation . . . . . . . . . . . . . . 31

Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

PharmacoScan™ Assay on the Biomek FXP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Before using the Biomek workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Breaking the light curtain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Pipette tip usage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Setting method preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Equipment, consumables, labware, and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Labware and materials used on the Biomek workstation deck . . . . . . . . . . . . . . . . . . . . . 41

Proper tray alignment and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Stain trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Loading tray consumables onto the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . 57

Reagent block template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Reservoir stickers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Related Biomek FXP Target Prep Express documentation . . . . . . . . . . . . . . . . . . . . . . . . 61

CHAPTER 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

PharmacoScan™ Assay on the Biomek FXP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Stage 1A: Multiplex PCR (mPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Input required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Equipment, consumables and reagents required. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

1: Prepare for mPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

2: Prepare the mPCR master mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

3: Set up the mPCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

4: Run the PharmacoScan mPCR thermal cycler protocol and proceed. . . . . . . . . . . . . . 67

5: Store the mPCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

4 PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP (Windows® 7) User Guide

Contents

Stage 1B: DNA amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Equipment, consumables, labware and reagents required . . . . . . . . . . . . . . . . . . . . . . . . 69

1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

2. Thaw and prepare the reagents and Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

3. Run the DNA Amplification step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Summary of DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Stage 2: Fragmentation and purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Input required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81


1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

2. Thaw and prepare the amplified DNA samples and reagents . . . . . . . . . . . . . . . . . . . . 83

3: mPCR Spike-in to Amplification Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

4. Run the Fragmentation step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

4. Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Summary of Fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Stage 3: Centrifugation and drying pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Equipment and consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Stage 4: Resuspension, Hybridization Preparation, and QC . . . . . . . . . . . . . . . . . . . . . . . . . 96

Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96


1. Preparing frozen pellets and Axiom Resusp Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

2. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

3. Thaw and prepare the reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

4. Run the Resuspension, Hybridization Preparation, and QC step . . . . . . . . . . . . . . . . . 98

Summary of Resuspension and Hybridization Preparation . . . . . . . . . . . . . . . . . . . . . . . 105

Stage 5: Preparation for the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . 109

About Stage 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Duration of GeneTitan™ MC Instrument reagent preparation and sample preparation . . 113

Equipment and consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

2. Prepare the reagents for GeneTitan Reagent Tray Preparation . . . . . . . . . . . . . . . . . . 116

3. Prepare the Sample Plate (if stored at –20°C) and the array plate . . . . . . . . . . . . . . . . 118

4. Prepare the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

5. Run the Preparation for GeneTitan™ step. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays . . . . . . . . . . . . 126

6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays. . . . . . . . . . . . . . . . 127

PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP (Windows® 7) User Guide 5

Contents

6c. Complete Stage 4: Preparation for GeneTitan™ - Multiple Plate Workflow . . . . . . . . 127

Summary of Preparation for GeneTitan MC™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . 129

CHAPTER 5 Array processing with the GeneTitan™ MC Instrument. 134

Before using the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Proper tray alignment and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Stain trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Email and telephone notifications from the GeneTitan™ MC Instrument . . . . . . . . . . . . . 139

GeneTitan™ MC Instrument lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Setup options for array plate processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Aborting a process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Stage 1: Create and upload Batch Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Stage 2: Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Setup the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

Load Axiom™ array plate and hyb tray onto the GeneTitan™ MC Instrument . . . . . . . . . 150

Load a second Axiom™ array plate and hyb tray onto the GeneTitan™ MC Instrument. . 156

Status window prompts and actions required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Stage 3: Ligate, Wash, Stain, and Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Proper installation of the GeneTitan™ tray consumables . . . . . . . . . . . . . . . . . . . . . . . . . 161

Load trays onto the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Continuing the workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Shutting down the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

CHAPTER 6 Processing eight PharmacoScan™ array plates per week . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

Overview of the eight plate workflow of the PharmacoScan™ Assay on the Biomek FXP . . 171

Duration of assay steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Thawing frozen plates of amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Thawing plates with frozen pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Simultaneous eight plate workflow: Target preparation and array processing of the PharmacoScan™ Assay on the Biomek FXP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

Day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

Day 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Day 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

Day 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

Day 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193


Contents

CHAPTER 7 Processing three PharmacoScan™ array plates per week . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

Overview of the three plate workflow of the PharmacoScan™ Assay on the Biomek FXP . . 197

Duration of assay steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Thawing frozen plates of amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

Three plate workflow: Target preparation and array processing of the PharmacoScan™

Assay on the Biomek FXP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

Day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

Day 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Day 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

Day 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210

Day 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

CHAPTER 8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

Biomek FXP Target Prep Express . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

GeneTitan™ Multi-Channel Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

Miscellaneous messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

Fluidic diagnostic messages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

Wash/Scan Resume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

Aborting a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

APPENDIX A Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

General safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

APPENDIX B Fragmentation quality control gel protocol . . . . . . . . . 226

Protocol for running a fragmentation quality control gel . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

E-Gels and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

Diluting the TrackIt™ Cyan/Orange Loading Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

Fragmentation QC gel protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227


Contents

APPENDIX C Sample quantitation after resuspension . . . . . . . . . . . 229

Protocol for sample quantitation after resuspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229


Quantitate the diluted samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

Assess the OD readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

Suggested protocol for OD quantitation using the DTX 880 . . . . . . . . . . . . . . . . . . . . . . . . 231

If performing sample quantitation on a plate reader other than the DTX880 . . . . . . . . . . . 237

APPENDIX D Registering samples in GeneChip™ Command Console™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

Creating a GeneTitan™ Array Plate Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

APPENDIX E Deionization procedure for GeneTitan™ trays and covers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

Deionization procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

Ion-indicator cap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

APPENDIX F GeneTitan™ Multi-Channel Instrument Care . . . . . . . . 245

Cleaning and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

Monthly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

Every six months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

Servicing the outer enclosure fan filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

Cleaning schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

Cleaning procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

Replacing the bottle filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

Removing and inspecting the filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

Replacing the filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

Replacing the xenon lamp in the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . 249

Lamp life/imaging device status notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Removing the xenon lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

Replacing the lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

Resetting the lamp counter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

Log files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

AGCC log files for GeneTitan™ MC Instrument Systems . . . . . . . . . . . . . . . . . . . . . . . . . 255

Insufficient disk space notice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256


Contents

APPENDIX G mPCR quality control gel protocol . . . . . . . . . . . . . . . 257

Protocol for running an mPCR quality control gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257


E-Gels and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

Prepare NEB 50 bp DNA ladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

Preparing mPCR samples for gel analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

mPCR QC gel protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264


1 The PharmacoScan™ Assay 96-Array Format Automated Protocol

Overview

Developed in collaboration with experts across the field of pharmacogenomics, PharmacoScan™ Solution is the industry’s broadest content genetic analysis system specifically designed to provide insight into the absorption, distribution, metabolism, and excretion (ADME) and transport of commonly prescribed medicines. By interrogating more than 4,600 markers in nearly 1,200 genes known to play a role in drug metabolism, traditional clinical researchers will gain unprecedented understanding into an individual’s ability to process those drugs with high evidence for genetic association, as well as those markers where moderate, low, preliminary and unknown evidence exists.

PharmacoScan Solution utilizes the same chemistry from the Axiom™ assay and GeneTitan™ Multi-Channel Instrument, a system that is preferred worldwide by genetic researchers for the efficient workflow, high-throughput, economic pricing, and reproducibility of results critical to multi-year data collection and analysis efforts.

Introduction

PharmacoScan™ Assay 96-Array Format Automated Protocol is available as a bundled kit that includes the arrays, reagents and consumables needed for processing one 96-format plate, each having 94 samples and 2 controls. The automated protocol described in this user guide leverages the Axiom 2.0 Assay method on the Biomek FXP for target preparation and GeneTitan reagent preparation. The multiplex PCR (mPCR) and mPCR spike-in steps are not part of the automated method and must be executed off-deck, manually.

PharmacoScan interrogates biallelic as well as multiallelic SNPs, indels and copy number variation (CNV) in a single assay workflow. Starting with genomic DNA, the samples are processed by performing an automated target preparation protocol followed by automated processing of the array plates on the GeneTitan MC Instrument.

• Target preparation uses methods including DNA amplification, fragmentation, purification and resuspension of the target in hybridization cocktail.

• The hyb-ready targets are then transferred to the Applied Biosystems GeneTitan™ Multi-Channel (MC) Instrument for automated, hands-free processing including hybridization, staining, washing and imaging.

PharmacoScan provides pharmacogenomic variation information for more than 4,600 ADME markers in nearly 1,200 genes. This content is sourced from globally endorsed consortium databases including, but not limited to CPIC, PharmGKB, and PharmaADME. Also included on PharmacoScan are high value markers for human leukocyte antigen (HLA) imputation, markers for killer cell immunoglobulin-like receptors (KIR), markers for human ancestry identification (AIM), a marker GWAS


Chapter 1 The PharmacoScan™ Assay 96-Array Format Automated ProtocolPharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP 1

backbone, and markers for sample ID and tracking. The combination of these high value markers, in addition to PharmacoScan’s ability to precisely call variants in critical genes on a microarray, compliments Thermo Fisher Scientific’s current solutions for pharmacogenomics using OpenArray™ Real-Time PCR and Ion Ampliseq™ NGS panels.

PharmacoScan is a multiplex genotyping assay which combines the proven Axiom chemistry with the incorporation of a multiplex PCR step to overcome some of the complexities associated with genotyping highly homologous markers. PharmacoScan software and algorithm developments include an allele translation and phenotyping tool and copy number aware genotyping. Array plates are processed on a GeneTitan™ MC Instrument controlled by Applied Biosystems™ GeneChip™ Command Console™

4.3 or higher. The resulting CEL files are analyzed by Axiom™ Analysis Suite 3.0 or higher, or by Applied Biosystems Microarray Power Tools 1.19 or newer.

For further information, please refer to ʺRelated documentationʺ on page 260.

The PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP must only be executed on controllers using the Windows 7 operating system.

PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP

Running the PharmacoScan Assay requires the following sets of steps:

1. Genomic DNA Prep -- Resulting in samples that meet requirements spelled out in Chapter 2, ʺGenomic DNA preparation and requirementsʺ on page 14.

2. A manual preparation of a multiplex PCR step (mPCR) followed by automated target preparation of the samples (see Chapter 4, ʺMultiplex PCR and Target preparation on the Biomek FXP with Windows® 7ʺ on page 62).

3. Target Prep of the samples: Chapter 4, ̋ Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7ʺ on page 62.

4. Array Processing, done with:

• GeneTitan MC Instrument

• GeneTitan Instrument Control software

• Applied Biosystems™ GeneChip™ Command Console Portal software

See Chapter 5, ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 134.

A list of the required equipment and supplies for running the PharmacoScan Assay using the Biomek FXP for automated target preparation can be found in the PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP Site Preparation Guide, Pub. No. 703473.


Chapter 1 The PharmacoScan™ Assay 96-Array Format Automated ProtocolPharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP1

Figure 1 PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman® Biomek FXP

10 μL

10 μL/well

20 μL/well

AMP Plate + mPCR

Pharmacoscan Assay Overview – Page 1 of 1

Stage 1A: mPCRDuration: ~ 3.5H

Multiplex PCR Thermal Cycler Program

of mPCR PLATE

Stage 1B: DNA AmplificationDuration: 23H

37°C incubation of AMPLIFICATION PLATE

DAY

1

DAY

2

Stage 2: Fragmentation and

Purification

DAY

3

Duration: 16-24H –20°C incubation of

PRECIPITATION PLATE

Stage 3: Centrifugation and

drying pellets

Duration: 23.5-24H Array Hybridization

DAY

4

Stage 5:Preparation for the

GeneTitanTM MC Instrument

Duration: ~ 12H Fluidics: 4.5H Scan: ~7.5H

mPCR Spike-Ininto AMP plate

Possible stopping point

mPCR Plate

gDNA Plate Setup5 ng/μL of gDNA

dddddd

Automated

Stage 1B: DNA AmplificationStage 1A: mPCR

Manual

Stage 4: Resuspension, Hybridization

Preparation, and QC


Chapter 1 The PharmacoScan™ Assay 96-Array Format Automated ProtocolPharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP 1

Running multiple plate workflows

Thermo Fisher Scientific provides workflows that allow you to run a set of samples and array plates through the protocol using a minimum of personnel and a forty-hour week. The timing of steps is critical because of the following constraints:

• Incubation after DNA Amplification is 23 hours.

• Hybridization in the GeneTitan MC Instrument is 23.5 hours.

• Reagent trays for wash/stain/imaging must be prepared as Hybridization finishes.

• Limits to when a second hyb tray and array plate can be loaded into the GeneTitan MC Instrument.

These limitations require careful timing. The details are covered in:

• Chapter 6, ʺProcessing eight PharmacoScan™ array plates per weekʺ on page 171.

• Chapter 7, ʺProcessing three PharmacoScan™ array plates per weekʺ on page 196.


2 Genomic DNA preparation andrequirements

Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Sources of genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

General requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Genomic DNA extraction/purification methods. . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Genomic DNA cleanup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Genomic DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Overview

The general requirements for genomic DNA (gDNA) sources and extraction methods are described in this chapter. The success of this assay requires uniform amplification of the genome starting with relatively intact gDNA. To achieve this, the gDNA must be of high quality, and must be free of contaminants that may affect the enzymatic reactions to be performed.

For this protocol, you will use the PharmacoScan™ Reagent Kit 96 Reactions (Cat. No. 913025). The kit contains two Control gDNAs, Control DNA 1 and Control DNA 2. This DNA meets the requirements outlined below, and both Control DNAs must be included on every plate for data analysis purposes. The size and purity of sample gDNA can be compared with those of the control DNA to assess sample quality.

Assay performance may vary for gDNA samples that do not meet the general requirements described below. However, the reliability of any given result should be assessed in the context of overall experimental design and goals.

Sources of genomic DNA

The following sources of human gDNA have been successfully tested in the PharmacoScan Assay 96-Array Format Automated Protocol for Biomek FXP with DNA that meets the above requirements.

• Blood

• Cell line

Other sample types have not been validated in this assay and are not currently supported.

Note: DNA derived from Formalin-Fixed Paraffin-Embedded (FFPE) blocks should not be used with this assay.


Chapter 2 Genomic DNA preparation and requirementsGeneral requirements 2

General requirements

• Starting DNA must be double-stranded for the purpose of accurate concentration determination.

• DNA must be of high purity.DNA should be free of DNA polymerase inhibitors. Examples of inhibitors include high concentrations of heme (from blood) and high concentrations of chelating agents (i.e., EDTA). The gDNA extraction/ purification method should render DNA that is generally salt-free because high concentrations of particular salts can also inhibit enzyme reactions. DNA purity is indicated by OD260/OD280

and OD260/OD230 ratios. The OD260/OD280 ratio should be between 1.8 and 2.0 and the OD260/OD230 ratio should be greater than 1.5. We recommend that DNA samples that do not meet these criteria be cleaned up as described under ʺGenomic DNA cleanupʺ on page 17.

• DNA must not be degraded.The approximate average size of gDNA may be assessed on a 1% agarose gel using an appropriate size standard control. Approximately 90% of the DNA must be greater than 10 Kb in size. Control DNA can be run on the same gel for side-by-side comparison.

Special requirements

Pre-amplification areaPrecautions are required when manipulating genomic DNA to avoid contamination with foreign DNA amplified in other reactions and procedures. It is recommended that genomic DNA manipulations are performed in a dedicated pre-amplification room or area separate from the main laboratory.

This pre-amplification area should have a dedicated set of pipettes and plasticware. If no dedicated area is available, use of a dedicated bench or a dedicated biosafety hood and dedicated pipettes is suggested. If no dedicated bench or biosafety hood is available, a set of dedicated pipettes is recommended.

Assessing the quality of genomic DNA using 1% Agarose E-gels

We recommend this quality control step to assess the quality of the gDNA prior to starting the assay.

Equipment and reagents recommended

Table 1 E-Gel® and reagents required

Item Supplier Part number

Mother E-Base Device

Thermo Fisher Scientific

EB-M03

Daughter E-Base Device EB-D03

E-Gel® 48 1% agarose gels G8008-01

RediLoad™ 750026

E-Gel® 96 High Range DNA Marker 12352-019


Chapter 2 Genomic DNA preparation and requirementsGeneral requirements2

Guidelines for preparing the Genomic DNA Plate for gel analysis• Loading a DNA mass of 10 ng to 20 ng per well is recommended. If lower amounts

are loaded, omission of the loading dye is recommended in order to improve visualization. Loading 25 ng gDNA per well can improve the image.

• Add 3 µL of 0.1X of RediLoad dye to each sample.

• Bring each sample to a total volume of 20 µL using H2O (for example, if the volume of genomic DNA is 5 µL, add 3 µL of RediLoad, and bring to 20 µL total by adding 12 µL of H2O).

• Seal, vortex and spin.

To run a 48-lane 1% agarose e-gel:

1. Power on for E-Base (red light).

2. Push the Power/Prg button to make sure the program is at EG mode (not EP).

3. Insert the two 48 well 1% agarose e-gels into the slots.

4. Remove 2 combs.

5. Load 20 µL from the above plate onto two 48 well 1% agarose e-gels.

6. Load 15 µL of diluted High Range DNA Marker (1:3 dilution or ~0.34 X from stock) into all marker wells (as needed).

7. Fill all empty wells with water.

8. Adjust the run time to ~27 min.

9. Push the Power/Prg button again (it will change from red to green).

When run time is reached (the ladder band reaches the end of the lane), the system will automatically shut off. The gel is then ready for imaging.

Figure 2 shows gel images of intact gDNA (that is suitable for use in the PharmacoScan™ Assay) and degraded gDNA samples. Customers whose gDNA is degraded (similar to the image in Figure 2) should perform a test experiment to investigate the performance of their samples in the PharmacoScan Assay prior to beginning any large scale genotyping projects.

Figure 2 Gel images showing intact gDNA and degraded gDNA.


Chapter 2 Genomic DNA preparation and requirementsGenomic DNA extraction/purification methods 2

Genomic DNA extraction/purification methods

Genomic DNA extraction and purification methods that meet the general requirements outlined above should yield successful results. Methods that include boiling or strong denaturants are not acceptable because the DNA would be rendered single-stranded and can no longer be accurately quantitated using a PicoGreen-based assay.

Genomic DNA cleanup

If a gDNA preparation is suspected to contain inhibitors, the following cleanup procedure can be used:

1. Add 0.5 volumes of 7.5 M NH4OAc, 2.5 volumes of absolute ethanol (stored at –20°C), to gDNA.

2. Vortex and incubate at –20°C for 1 hr.

3. Centrifuge at 12,000 xg in a microcentrifuge at room temperature for 20 min.

4. Remove supernatant and wash pellet with 80% ethanol.

5. Centrifuge at 12,000 xg at room temperature for 5 min.

6. Remove the 80% ethanol and repeat the 80% ethanol wash one more time.

7. Resuspend the pellet in reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA).

(See the PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP Site Preparation Guide, Pub. No. 703473 for reagents, equipment, labware and consumables for Axiom 2.0 Assay).

Genomic DNA preparation

This step needs to be done before proceeding with the mPCR and DNA amplification stages. The genomic DNA (gDNA) you will process using the PharmacoScan Assay 96-Array Format Automated Protocol for Biomek FXP should meet the general requirements listed earlier in this chapter. The amount of gDNA is 50 ng for the mPCR step and 100 ng for the PharmacoScan whole-genome amplification step.

To prepare gDNA:

ʺ1. Thaw samples and controlʺ

ʺ2. Quantitate and dilute test sample gDNAʺ

ʺ3. Aliquot the diluted samples and the controls DNA 1 and DNA 2ʺ

ʺ4. Freeze or proceedʺ

ʺ5. Create a Batch Registration fileʺ

Table 2 Input requirements for PharmacoScan Assay 96-Array Format Automated Protocol for Biomek FXP

Assay step Volume per well Input mass per well gDNA concentration

Stage 1A: mPCR 10 μL 50 ng 5 ng/μL

Stage 1B: DNA Amplification 20 μL 100 ng 5 ng/μL


Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation2

Duration Thirty minutes to an hour for reagents to thaw and half an hour for setup.

Equipment, consumables and reagents required

Equipment and consumablesThe equipment and consumables listed in Table 3 are required for this stage.

ReagentsThe reagents listed in Table 4 are required for this stage.

Table 3 Equipment and consumables required for genomic DNA preparation

Quantity Item

As required Adhesive seals for plates

1 Ice bucket, filled with ice

1 each Pipettes:• Single-channel P10 or P20 • Optional: multi-channel P10 or P20

As required Pipette tips

1 Plate, deep well: Beckman Deep Well Titer, polypropylene; Cat. No. 267007

1 96 well PCR plate (Bio-Rad HSS-9641 for Applied Biosystems 9700, Applied Biosystems Veriti™, Applied Biosystems ProFlex™, and Bio-Rad HSP-9631 for Eppendorf Master™ Cycler pro S)

1 Plate centrifuge

1 Plate spectrophotometer (required only if no OD measurements available for samples)

1 Vortexer

Table 4 Reagents required for Genomic DNA Preparation

Reagent Supplier Cat. No.

From the PharmacoScan Reagent Kit 96 Reactions

• Control DNA 1 and Control DNA 2 (PharmacoScan Module B)

Thermo Fisher

Scientific

Part. No. 912897

User-supplied

• Reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA)

Thermo Fisher

Scientific

75793

• Quant-iT™ PicoGreen™ dsDNA Assay Kit Life Technologies

P7589


Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation 2

1. Thaw samples and control

Thaw the components listed below to room temperature:

• gDNA samples

• Control DNA 1 and Control DNA 2 (from PharmacoScan Module B)

To thaw, either:

• Place items on benchtop for one hour

• Thaw in a water bath:

a. Fill a small plastic dish with Millipore water. Do not overfill as the level of the water should not overflow when the control DNA tubes or plates are placed in the bath.

b. Thaw the sealed Sample Plate and control DNA tubes for a half-hour.

c. Remove the Sample Plate and/or control DNA tube from the water bath and wipe-dry using lab wipes. Ensure the outside is completely dry before opening the Sample Plate or tube to minimize any contamination, which can lead to reaction failure.

2. Quantitate and dilute test sample gDNA

To quantitate and dilute test sample gDNA:

1. Gently vortex (50% maximum) and spin the gDNA.

2. Recommendation: quantitate each sample (e.g., using the Quant-iT™ PicoGreen® dsDNA Kit).

3. Using reduced EDTA TE buffer, dilute each sample to a concentration of 5 ng/µL.

4. Seal, vortex and spin.

Note: Do NOT dilute the Control DNA 1 or Control DNA 2 from PharmacoScan Module B (Part No. 912897). They are already at the working concentration.

Note: We strongly recommend you determine your sample concentrations using the Quant-iT PicoGreen assay by Life Technologies (Cat. No. P7589). Sample concentration determined by UV absorbance is often inaccurate and can yield different results.

3. Aliquot the diluted samples and the controls DNA 1 and DNA 2

Next, the samples and controls are placed in a deep well plate for DNA amplification and into a 96-well PCR plate for mPCR:

Note: Make sure gDNA is well mixed before plating.

Amplification sample plate• Beckman Deep Well Titer Plate; Cat. No. 267007

Aliquot diluted samples and controls to the deep well plate:

1. Aliquot 20 µL of each diluted gDNA sample to the Beckman Deep Well Titer Plate as shown in Figure 3.

2. Pipet 20 µL of Control DNA 1 to well G12 and 20 µL of Control DNA 2 to well H12.

3. Seal and spin.

IMPORTANT! Control DNA 1 and Control DNA 2 are required for assay performance. Both controls must be included on mPCR and Amplification Sample Plates and placed in indicated wells. Controls need to be run every time assay is performed.


Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation2

mPCR sample plate• Bio-Rad 96-well plate; HSS-9641 for Applied Biosystems 9700, Applied Biosystems Veriti, Applied Biosystems ProFlex, Bio-Rad 96 well plate; HSP-9631 for Eppendorf Mastercycler pro S

Aliquot diluted samples and controls to the mPCR sample plate:

1. Aliquot 10 µL of each diluted gDNA sample to the 96-well PCR plate as shown in Figure 3.

2. Pipet 10 µL of Control DNA 1 to well G12 and 10 µL of Control DNA 2 to well H12.

3. Seal and spin.

4. Freeze or proceed

At this point you can:

• Store the Sample Plate at –20°C, or

• Proceed to DNA Amplification for Automated Target Prep (see Chapter 4, ʺMultiplex PCR and Target preparation on the Biomek FXP with Windows® 7ʺ on page 62).

Note: You can leave the gDNA Sample Plate at room temperature if proceeding immediately to mPCR and DNA Amplification.

Figure 3 Aliquoting genomic DNA

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

C1

C2

Amplification Sample Plate mPCR Sample Plate

Beckman Deep Well Titer Plate20 μL/well

96 Well PCR Plate10 μL/well

C1 = Control DNA 1C2 = Control DNA 2


Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation 2

5. Create a Batch Registration file

GeneTitan Array Plate Registration files contain information that is critical for:

• Data file generation during imaging.

• Tracking the experimental results for each sample loaded onto an array plate.

Detailed instructions for creating this file are located in Appendix D, ʺRegistering samples in GeneChip™ Command Console™ʺ on page 238. See also Figure 4 for a screen shot showing an example of a batch registration file.

To create a Batch Registration file:

1. Open AGCC Portal Samples, and select:

a. GeneTitan Array Plate Registration.

b. The array plate format.

c. Click Download.

2. Enter a unique name for each sample and any additional information.

3. Save the file.

The array plate barcode will not be scanned until you are ready to load the array plate and samples onto the GeneTitan MC Instrument for processing.

IMPORTANT! It is very important to create and upload a GeneTitan™ Array Plate Registration file with your sample information prior to loading the array plate and hyb tray into the GeneTitan MC Instrument. We recommend that you create (but not upload) this file at the same time you prepare your plate of genomic DNA. When your samples are ready for hybridization, you will scan the array plate barcode and upload the file to Applied Biosystems™ GeneChip™ Command Console (AGCC).

Figure 4 Example of a Batch Registration file

Your specific information is populated here.


3 Preparation before you start

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Requirements and recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Equipment, consumables, and reagents required for mPCR preparation . . . . . 31

PharmacoScan™ Assay on the Biomek FXP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Before using the Biomek workstation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Equipment, consumables, labware, and reagents. . . . . . . . . . . . . . . . . . . . . . . . . . 39

Introduction

This PharmacoScan Assay format allows the user to run the PharmacoScan™ Assay for 96 samples once using one PharmacoScan™ Reagent Kit 96 Reactions (Cat. No. 913025) and one QIAGEN Multiplex PCR Plus Kit (QIAGEN Cat. No. 206152), which must be purchased separately. This section provides information on requirements, recommendations, procedures that are performed during manual preparation of the mPCR step and steps that are critical to the success of the automated target preparation on the Biomek FXP. It is essential that you familiarize yourself with the information in this section prior to running the PharmacoScan Assay.

A list of all equipment and resources required for the PharmacoScan Assay is in the PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman® Biomek FXP Site Preparation Guide, Pub. No. 703473.

PharmacoScan™ Assay arrays, reagents, and GeneTitan consumables required

The table below lists the PharmacoScan reagents and GeneTitan consumables required to process one PharmacoScan 96F Array Plate. The table also lists the QIAGEN Multiplex PCR kit required for the mPCR preparation portion of the PharmacoScan Assay.

Table 5 PharmacoScan™ Assay arrays, reagents, and GeneTitan consumables required

Quantity Description Supplier Cat. No

1 PharmacoScan™ 96F Array PlateThermo Fisher

Scientific

903160

1 Axiom™ GeneTitan™ Consumables Kit 901606

1 PharmacoScan™ Reagent Kit 96 Reactions 913025

1 QIAGEN Multiplex PCR Plus Kit, 100 Reactions QIAGEN 206152


Chapter 3 Preparation before you startPharmacoScan™ Assay arrays, reagents, and GeneTitan consumables required 3

Reagents required PharmacoScan™ Reagent Kit 96 Reactions, Cat. No. 913025The PharmacoScan Assay 96-Array Format Automated Protocol for Biomek FXP uses the PharmacoScan Reagent Kit 96 Reactions (Cat. No. 913025). Each kit consist of 6 modules for different stages of the assay with some modules having both 4°C and –20°C pouches. There are specific instructions for which reagents are needed and how to treat them within each stage.

Table 6 PharmacoScan™ Reagent Kit 96 Reactions, Cat. No. 9130251

Component Storage

Module 1: Part No. 901711• Axiom Denat Soln 10X• Axiom Neutral Soln • Axiom Water

• Axiom Amp Soln• Axiom Amp Enzyme

–25°C to –15°C

Module 2: Pouch 1 of 2: Part No. 901528• Axiom Frag Enzyme• Axiom 10X Frag Buffer• Axiom Precip Soln 2

• Axiom Hyb Buffer• Axiom Hyb Soln 1

–25°C to –15°C

Module 2: Pouch 2 of 2: Part No. 901529• Axiom Frag Diluent• Axiom Frag Rxn Stop• Axiom Precip Soln 1

• Axiom Resusp Buffer• Axiom Hyb Soln 2

2°C to 8°C

Module 3• Axiom Wash Buffer A: Part No. 901446

(2 bottles per kit)• Axiom Wash Buffer B: Part No. 901447

(1 bottles per kit)

• Axiom Water: Part No. 901578 (1 bottle per kit)

room temperature

Module 4: Pouch 1 of 2: Part No. 901278• Axiom Ligate Buffer• Axiom Ligate Enzyme• Axiom Ligate Soln 1

• Axiom Probe Mix 1• Axiom Stain Buffer• Axiom Stabilize Soln

–25°C to –15°C

Module 4: Pouch 2 of 2: Part No. 901276• Axiom Ligate Soln 2• Axiom Probe Mix 2• Axiom Wash A• Axiom Stain 1-A• Axiom Stain 1-B

• Axiom Stain 2-A• Axiom Stain 2-B• Axiom Stabilize Diluent• Axiom Water• Axiom Hold Buffer

2°C to 8°C

PharmacoScan Module A: Part No. 912896• 10X mPCR primers

–25°C to –15°C

PharmacoScan Module B: Part No. 912897• Control DNA 1 • Control DNA 2

–25°C to –15°C

1 Sufficient for processing one PharmacoScan 96-array format plate.


Chapter 3 Preparation before you startRequirements and recommendations3

QIAGEN Multiplex PCR Plus Kit (100), QIAGEN Cat No. 206152The QIAGEN Multiplex PCR Plus Kit should be stored at –30 to –15°C immediately upon receipt. For detailed information on this product, visit the supplierʹs website (www.qiagen.com).

Components included in the kit:

• 2x Multiplex PCR Master Mix (3 x 0.85 mL)

• 5x Q-Solution (1 x 2 mL)

• RNase-Free Water (2 x 1.9 mL)

• 10x CoralLoad® Dye (1 x 1.2 mL)**This component is not needed for the PharmacoScan Assay and can be discarded.

Requirements and recommendations

This section describes requirements and recommendations for facilities and equipment needed to perform the PharmacoScan Assay 96-Array Format Automated Protocol.

Room temperature When referred to in the PharmacoScan Assay 96-Array Format Automated Protocol, room temperature is 18 to 25°C.

Special requirements

Fume hoodAt certain steps in the protocol we recommend the use of adequate local or general ventilation to keep airborne concentrations low.

A fume hood is suggested as a way to achieve the desired concentration. Thus, a fume hood is strongly recommended for several steps of this assay.

Control requirementsA negative control is not required for this assay.

Two controls are required for proper data analysis. These controls, Control DNA 1 and Control DNA 2, are included in the PharmacoScan Reagent Kit, Module B (Part No. 912897).


Chapter 3 Preparation before you startRequirements and recommendations 3

Thermal cycler recommendations and protocols

We have verified the performance of this assay using the thermal cyclers listed below in their 96-well block configurations:

Table 7 Thermal cycler guidance

Thermal cycler program

Verified thermal cyclers PharmacoScan mPCR

PharmacoScan Denature

Biometra TRobot 96 (on-deck) No

Applied Biosystems 2720 No

BioRad/MJ DNA Engine Tetrad® 2 PTC-0240G No

Applied Biosystems 9700 (with a gold or silver block)

Applied Biosystems Veriti

Applied Biosystems ProFlex

Eppendorf® Mastercycler® pro S

IMPORTANT! Always use the heated lid option when programming protocols. The PharmacoScan mPCR protocol was validated using the “9600 mode” on the Applied Biosystems 9700, Applied Biosystems Veriti, and Applied Biosystems ProFlex thermal cyclers. The “Safe” mode was used for the Eppendorf Mastercycler pro S. Refer to the manufacturer’s user guide for programming information.

Figure 5 PharmacoScan mPCR thermal cycler protocol (Stage 1A)


Chapter 3 Preparation before you startRequirements and recommendations3

The mPCR step of the PharmacoScan Assay has been validated with the Applied Biosystems 9700 (with gold-plated or silver block) Applied Biosystems Veriti, Applied Biosystems ProFlex, and Eppendorf Mastercycler pro S. Use of other thermal cyclers for this stage may result in assay failure and may violate the array and reagent replacement policy.

Note: Two thermal cylers are required if running the three plate per week or the eight plate per week automated target preparation workflow.

PCR plate type by thermal cycler for mPCR

Table 8 provides details into the consumables to be used with each thermal cycler when executing the mPCR step.

Figure 6 PharmacoScan Denature thermal cycler protocol (Stage 4)

WARNING! Evaporation during denaturation can negatively impact assay performance. Use the recommended thermal cycler consumables and sealing film to eliminate condensation and evaporation. For thermal cyclers with variable lid tension (such as the Bio-Rad PTC-200 or Tetrad 0240) please follow the manufacturer’s instructions for adjusting lid tension.

Table 8 PCR plate type by thermal cycler for mPCR

Thermal cycler model

PCR plate type Seal1

Applied Biosystems 9700

• Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (P/N HSS-9641)

MicroAmp™ Clear Adhesive Film from Applied Biosystems (Cat. No. 4306311)

Applied Biosystems Veriti



Applied Biosystems ProFlex



Eppendorf Mastercycler pro S

• Bio-Rad Hard-Shell Low Profile 96-well Full-Skirt PCR Plate (P/N HSP-9631)


1 Microseal ‘B’ film from Bio-Rad (P/N MSB-1001) may be used in place of MicroAmp Clear Adhesive Film for the Applied Biosystemsthermal cycler.


Chapter 3 Preparation before you startRequirements and recommendations 3

Oven recommendations

The following ovens are recommended:

• BINDER ED 56 Drying and Heating Chamber. Refer to the PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP Site Preparation Guide, Pub. No. 703473, for ordering information.

• Applied Biosystems GeneChip Hyb Oven 645

Note: The GeneChip™ Hybridization Oven 640 is currently not supported with the PharmacoScan Assay; however, if you want to utilize it in the workflow please contact your Field Service Engineer (FSE) or Technical Support regarding the compatibility of this oven with the PharmacoScan Assay 96-Array Format Automated Protocol.

– If using a GeneChip Hyb Oven, set the rotation speed to 15 RPM to aid in even heat distribution.

– For either GeneChip Hyb Oven, plates are placed in the bottom of the oven. To avoid interfering with the rotation apparatus, do not stack plates in the oven.

Plate centrifuge One plate centrifuge is required for the PharmacoScan Assay. Refer to the PharmacoScan Assay 96-Array Format Automated Protocol for Biomek FXP Site Preparation Guide, Pub. No. 703473, for an appropriate plate centrifuge that can be used. When centrifuging and drying pellets as instructed under ʺStage 3: Centrifugation and drying pelletsʺ on page 94, the centrifuge must be able to spin down plates at:

• Rcf: 3200 xg (4000 RPM for the Eppendorf 5810R with the rotor configuration described in the PharmacoScan Assay 96-Array Format Automated Protocol for Biomek FXP Site Preparation Guide, Pub. No. 703473).

• Temperature: 4°C and room temperature.

In addition, the bottom of the rotor buckets should be soft rubber to ensure that the deep-well plates do not crack. Do not spin plates in metal or hard plastic buckets.

Plate shakers We recommend using one of the following shakers listed in Table 9.

Equipment care and calibration

Lab instrumentation plays an important role in the successful completion of this assay. To aid in maintaining consistency across samples and operators, all equipment must be regularly calibrated and well maintained, including:

• All pipettes, thermal cyclers, and ovens

• Plate spectrophotometer

Table 9 Shakers

Shaker Supplier Cat. No.

Thermo Scientific™ Compact Digital Microplate Shaker

Thermo Scientific 88880023

Jitterbug™ Boekel Scientific Model 130 000


Chapter 3 Preparation before you startProcedures3

Procedures

This section covers procedures you may need to do repeatedly during the workflow, or which are critical to the performance of the assay.

Seal, vortex, and spin

Unless otherwise stated in the protocol, follow the guidelines below when instructed to seal, vortex, and spin plates or reagent tubes for the mPCR preparation and the Biomek FXP Target Prep Express portion of this assay:

• Seal plates—we recommend using MicroAmp Clear Adhesive Films to seal your plates.

Blot-dry—Prior to sealing plates, we recommend checking the top of the plate to make sure that there are no droplets. If droplets are present, blot-dry the top of the plate before sealing to ensure a tight seal.

a. To remove droplets prior to sealing overlay a sheet of Kimwipe across the top of the plate and gently pat down to dry.

b. Lift the sheet off the plate and discard. Confirm the top of the plate is dry and seal the plate as usual.

• Vortex:

– Plates:

• For deep well plates (such as Beckman Deep Well Titer Plate), vortex at maximum speed for 5 seconds in each sector for a total of 5 sectors (Figure 7).

• Plates 1 sec each corner, and 1 sec in the center at the maximum setting (Figure 7).

– Reagent Vials: 3 times at maximum speed, 1 sec each time.

Note: In the procedures, “vortex twice” means to repeat the vortexing step.

IMPORTANT! Always ensure that your plates are tightly sealed. A tight seal will prevent sample loss and cross-well contamination, particularly when plates are being vortexed. NEVER REUSE A SEAL. ALWAYS USE A NEW SEAL.

Figure 7 Vortex plates at the corners and center


Chapter 3 Preparation before you startProcedures 3

• Spin—when instructed to spin plates or reagent vials, follow these guidelines unless otherwise instructed (for example, when centrifuging and drying pellets, see Step 2 in the section ʺStage 3: Centrifugation and drying pelletsʺ on page 94).

– Plates:

• Spin at 1000 rpm for 30 sec at room temperature.

• Do not spin for more than 1 min.

– Reagent Vials: 3 sec.

About the reagents and master mix preparation

PharmacoScan Reagent Kit 96 Reactions components • Caps on the vials are color-coded by assay stage.

• Properly store all enzyme reagents, especially enzyme-containing vials. Improper storage methods can profoundly impact activity.

Reagents from other suppliers• Use only fresh reagents from the recommended suppliers to help eliminate

changes in pH or the salt concentration of buffers.

• Consult the appropriate MSDS for reagent storage and handling requirements.

mPCR Master Mix preparation• Carefully follow the master mix recipe. Use pipettes that have been calibrated to

± 5%.

• If you run out of master mix during the procedure, a volume error has been made, or the pipettes are not accurate; we recommend that you stop and repeat the experiment.

When using reagents at the lab bench• Properly chill essential equipment, such as reagent coolers, before use.

• Ensure that enzymes are kept at –20°C until needed. When removed from the freezer, immediately place in a cooler that has been chilled to –20°C.

IMPORTANT! The PharmacoScan Assay 96-Array Format Automated Protocol is compatible only with reagents from the PharmacoScan Reagent Kit 96 Reactions (Cat. No. 913025). These reagents are not interchangeable with reagents from other reagent kits, such as CytoScan™, SNP 6.0, DMET Plus, etc.


Chapter 3 Preparation before you startProcedures3

Pipettes and pipetting

To efficiently process samples:

• Use a pipette of appropriate size for the volume of liquid being transferred (Table 10).

• We recommend the use of Rainin pipettes and tips. Thermo Fisher Scientific has only verified the use of Rainin multi-channel pipettes in this assay. The use of other pipettes may impact the timing of the protocol and may adversely impact the assay. Pipette substitution may violate the terms of the PharmacoScan Assay 96-Array Format Automated Protocol and array replacement policy.

• Always use pipettes that have been calibrated.

• It is essential that you be proficient with the use of single- and multi-channel pipettes. To familiarize yourself with the use of multi-channel pipettes, we strongly recommend practicing several times before processing actual samples. Use water and reagent reservoirs to get a feel for aspirating and dispensing solutions to multiple wells simultaneously.

Single-channel pipettes and serological pipettesUse single-channel pipettes for preparing Master Mixes and for puncturing bubbles in GeneTitan trays. The single-channel pipettes will not be used for working with the plates or trays otherwise.

• Use single channel pipettes for volumes less than or equal to 2 mL. For volumes between 1 and 2 mL, add the reagent in two portions with a fresh tip for each portion.

• Use serological pipette for volumes 2 mL.

Multi-channel pipettesUse 8 or 12-channel pipettes when working to add Master Mix or to transfer samples to plates.

• Use a pipette of appropriate size for the volume of liquid being transferred.

• Change pipette tips after each transfer or addition.

Matrix™ 25 mL reagent reservoirs

The mPCR preparation step of the PharmacoScan Assay requires the use of disposable reservoirs with a “trough within a trough” design. This special design maximizes the amount of liquid accessible to pipette tips when using small amounts of reagent.

Table 10 Recommended pipette sizes

Pipette size Recommended volume range

Single channel P20 / 12-channel P20 1-20 μL



Figure 8 Dispense reagents from Matrix™ 25 mL reagent reservoirs


Chapter 3 Preparation before you startEquipment, consumables, and reagents required for mPCR preparation 3

Equipment, consumables, and reagents required for mPCR preparation

Equipment required Thermal cyclerRefer to ʺThermal cycler recommendations and protocolsʺ on page 25

Plate centrifugeRefer to ʺPlate centrifugeʺ on page 27

Consumables required

Table 11 Consumables required for mPCR preparation

Labware Supplier and Cat. No.

Image

Bio-Rad® Hard-Shell® 96-well plate

Bio-Rad Hard-Shell Low-Profile 96-Well Skirted PCR Plates

Note: Please refer to Table 8 for the PCR plate type recommended for your specific thermal cycler.

Bio-Rad

Cat. No. HSP-9631

96 Half-Skirt Plate

Bio-Rad Hard-Shell® High-Profile 96-Well Semi-Skirted PCR Plates

Note: Please refer to Table 8 for the PCR plate type recommended for your specific thermal cycler.

BioRad

Cat. No. HSS-9641


Chapter 3 Preparation before you startEquipment, consumables, and reagents required for mPCR preparation3

Reagents required PharmacoScan Module A (Cat. No. 912896), part of the PharmacoScan Reagent Kit 96 Reactions (Cat. No. 913025)The mPCR preparation step only requires the mPCR Primers in Module A. Refer to Table 6 on page 23 for the complete kit description.

15 mL conical-bottom centrifuge tube, Polypropylene

Various

96-well Block

Cooling Chamber for 0.2 mL tubes, 96 holes (4 for 1.5 mL & 6 for 0.5 mL tubes), Dim.: 6 1/8”L x 3 1/8”W x 1” H

Diversified Biotech

Cat. No. CHAM-1000

Thermo Scientific™ Matrix™ Reagent Reservoirs, 25 mL

Thermo ScientificCat. No. 8093-11

Table 11 Consumables required for mPCR preparation (Continued)

Labware Supplier and Cat. No.

Image


Chapter 3 Preparation before you startPharmacoScan™ Assay on the Biomek FXP 3

QIAGEN Multiplex PCR Plus Kit (100) (QIAGEN Cat. No. 206152)QIAGEN Multiplex PCR Plus Kit (QIAGEN Cat. No. 206152) is used with PharmacoScan Reagent Kit 96 reactions to process 96 samples. The QIAGEN kit configuration is as follows:

• 3 tubes of 0.85 mL of Multiplex PCR Master Mix, 2X

• 1 tube of 2 mL of Q-Solution, 5X

• 2 tubes of 1.9 mL of RNase-free Water

• 1 tube of 1.2 mL of CoralLoad® Dye, 10X

Note: The CoralLoad Dye will not be needed for PharmacoScan Assay 96-Array Format Automated Protocol and can be discarded. All three tubes of 2x Multiplex PCR Master Mix will be needed to process one PharmacoScan 96F array plate, but only one tube of Water and one tube of Q-Solution is required.

PharmacoScan™ Assay on the Biomek FXP

The PharmacoScan Assay 96-Array Format Automated Protocol leverages the Axiom 2.0 Assay method on the Biomek FXP for target preparation and GeneTitan reagent preparation to process 96 samples at a time. Please note that the multiplex PCR (mPCR) and mPCR spike-in steps are not part of the automated method and is executed off-deck, manually. The protocol is performed in two parts:

• Part 1: Target Preparation

– mPCR preparation and mPCR spike-in steps are performed manually, off-deck.

– All other steps are performed on the Biomek FXP Target Prep Express

• Part 2: Array Processing is performed on the GeneTitan™ Multi-Channel (MC) Instrument

A list of all equipment and resources for the PharmacoScan Assay with Automated Target Prep is in the PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP Site Preparation Guide, Pub. No. 703473.

This chapter includes instructions for Part 1: Target Preparation. These instructions are presented as follows:

• ʺBefore using the Biomek workstationʺ on page 34

– ʺBreaking the light curtainʺ on page 34

– ʺPipette tip usageʺ on page 35

– ʺSetting method preferencesʺ on page 35

– ʺEquipment, consumables, labware, and reagentsʺ on page 39

– ʺReagent block templateʺ on page 59

– ʺRelated Biomek FXP Target Prep Express documentationʺ on page 61

Using the automated target preparation protocol, eight array plates can be processed per work week for a total of 768 arrays, 752 samples and 16 controls. See Chapter 6, ʺProcessing eight PharmacoScan™ array plates per weekʺ on page 171 for further information.

IMPORTANT! Before proceeding to mPCR and DNA Amplification, do the gDNA preparation described in Chapter 2, ̋ Genomic DNA preparation and requirementsʺ on page 14.


Chapter 3 Preparation before you startBefore using the Biomek workstation3

Before using the Biomek workstation

Breaking the light curtain

For your safety, the Biomek FXP Target Prep Express is designed to immediately halt all movement when the light curtain is broken.

Light curtain broken while running the assayFor your safety, the Biomek FXP Target Prep Express is equipped with a light curtain (Figure 9). The light curtain senses when an object (such as a hand or an arm) enters the space surrounding the deck. When this curtain is broken, all movement on the deck halts until the user either clicks OK to resume the activity that was taking place, or aborts the activity. Incubation timers are not interrupted.

Figure 9 Prompt displayed when the light curtain is broken during the process referred to as Homing All Axes.

The light curtain covers the front of the instrument. The sides are protected by Plexiglas. Both areas are shown as red in this figure. Any penetration of the light curtain from outside the deck halts all movement of the workstation. To resume activity on the deck, click OK. To abort the step or other activity, click Stop on the toolbar.


Chapter 3 Preparation before you startBefore using the Biomek workstation 3

Pipette tip usage

Setting method preferences

Typically you will set the method preferences for the Biomek software once. The settings you select will:

• Determine which process controls will be run during stages 2 and 3 (Fragmentation and Resuspension). The process controls include:

– Highly recommended: Preparation of sample dilution plates for OD and gel analysis during Resuspension and Hyb Prep. The dilution plates are taken off-deck. One is used for OD quantitation to evaluate DNA mass; the other is used to check fragment size.

– If desired: Prompt you to manually take samples for DNA quantitation prior to fragmentation.

• Select deck configuration options for the thermal cycler (choose between having an integrated Biometra TRobot 96 thermal cycler or no integrated thermal cycler).

Table 12 Pipette tip usage for one full run of the Axiom™ 2.0 Assay on the Biomek workstation

StepMulti-Channel

P50, Pink96 tips, Cat. No. A21586

Multi-Channel AP96 P250,

Aqua96 tips,

Cat. No. 717253

Span-8 Span P250,

Green96 tips,

Cat. No. 379503

Span-8 Span P1000,

Yellow96 tips,

Cat. No. 987925

96 rxns 96 rxns 96 rxns 96 rxns

DNA Amplification — 96 tips — 33 tips

Fragmentation — 96 tips 8 tips 36 tips

Resuspension and Hybridization Preparation 96 tips 96 tips 17 tips 23 tips

Preparation for the GeneTitan™ MC Instrument• Denature samples• Transfer denatured samples to hyb tray• Prepare GeneTitan™ reagent trays

— 96 tips 26 tips 96 tips

TOTAL 96 tips 384 tips 51 tips 188 tips



To set the method preferences:

1. Launch the Biomek Software.

2. Open Project Open Project Axiom 2.0 Target Prep and click OK (Figure 10).

3. Open File Open to display the Open Method window (Figure 11).

Or click the Open Method icon

4. Select Axiom 2.0 Target Prep and click OK.

.

5. In the left pane of the window, select Axiom Target Prep (Figure 12).

Figure 10 Opening the Axiom 2.0 Target Prep Project

Figure 11 Opening the Axiom Target Preparation method

The Axiom 2.0 Target Prep project folder must be displayed in this menu.


Chapter 3 Preparation before you startBefore using the Biomek workstation 3

The method preferences selection window is displayed on the right.

6. Set Method Preferences:

Device Configuration Option— these settings can be changed at the start of each step

• What type of thermal cycler setup do you have?

• TRobot-select this option to perform the denaturation of the Axiom Hyb Ready Plate on the integrated Biometra TRobot 96 thermal cycler

• No integrated thermal cycler - select this option to perform the denaturation of the Axiom Hyb Ready Plate on an off-deck thermal cycler or if your Biomek does not have an integrated thermal cycler. A list of thermal cyclers that have been verified with the assay can be found below. When selecting this option, select the appropriate plate type that should be used for the Hyb Ready Plate.

– Select the Bio-Rad Skirted option when using the HSP-9631 plate for the PTC-200 or the Bio-Rad Tetrad 0240G thermal cycler.

Figure 12 Click Startup Dialog to open the Method Preferences Selection window

The selections made in this box, Device Configuration Option, are displayed each time this window appears.

The options in the Custom Run Options box must be selected prior to starting a run.

You are not prompted to select any preferences at start up.



– Select the Bio-Rad Semi-Skirted on Costar Round U-Bottom option for the Applied Biosystems 9700 or the Applied Biosystems 2720 thermal cycler. The Bio-Rad HSS-9601 plate must be stacked onto the Costar Round Bottom plate from Corning (VWR International Cat. No. 29442-392, E&K Scientific: EK 680568, Corning Mfg. Cat. No. 3795) for the Biomek FXP Target Prep Express to prepare the Hyb Ready Plate.

We have verified the performance of this assay using the Biometra TRobot 96 on the Beckman Biomek Target Prep Express liquid handler. We have also verified the performance of this assay using the following off-deck thermal cyclers, with 96-well blocks:

• Bio-Rad PTC-200G

• Applied Biosystems 9700 with a gold, or silver block

• Applied Biosystems 2720

• Bio-Rad / MJ Tetrad® 2 PTC-0240G

• Applied Biosystems Veriti

• Applied Biosystems ProFlex

• Eppendorf® Mastercycler® pro S

The performance of this assay has not been verified with other thermal cyclers.

Use of other thermal cyclers may result in assay failure and may violate the Axiom Array and Reagent replacement policy.

The thermocycler needs to be programmed with the “Axiom Denature” protocol:

a. 95°C 10 min

b. 48°C 3 min

c. 48°C hold

Use the heated lid option when setting up or running the protocol.

Custom Run Options— These settings are displayed in this window only. You must select/deselect here.

• QC check points

• Prompt for manual DNA quantitation before fragmentation—the workstation will pause following inactivation of the DNA amplification reaction to allow you to manually remove an aliquot of each sample for off-line (manual) DNA quantitation. This extra quality control step is available for troubleshooting the DNA amplification reaction.

• Prepare plates for gel QC and OD after hyb rxn transfer— the workstation will prepare two plates of resuspended samples properly diluted for the fragmentation gel QC and OD quantitation process control checks. See Appendix B, ̋ Fragmentation quality control gel protocolʺ on page 226 and Appendix C, ʺSample quantitation after resuspensionʺ on page 229 for instructions and result assessment guidelines.

WARNING! Evaporation during denaturation can negatively impact assay performance. Use the recommended thermal cycler consumables and sealing film to eliminate condensation and evaporation. The arched, auto-sealing metal plate with P pads as shown in Table 18 on page 41 should be replaced as per the manufacturer’s recommendation. If using the TRobot, complete the lid setting per the manufacturerʹs instructions.


Chapter 3 Preparation before you startEquipment, consumables, labware, and reagents 3

• Run method in test mode—select this option to skip all of the incubation timers in a step. If selected, a prompt is displayed asking you to confirm that you want to run a step in test mode (Figure 12). Use this option to perform runs using deionized water only, not actual reagents or samples.

7. Click Start at the top of the left pane to close the default settings window.

Equipment, consumables, labware, and reagents

Thermo Fisher Scientific now offers labware consumables kits for target preparation:

• Axiom 96 Consumable Kit for Biomek FXP (Sufficient for 4 x 96 rxn runs,Cat. No. 902800)

• Axiom 96 Consumable Kit for QC (Sufficient for 40 x 96 rxn runs, Cat. No. 902801)

• Axiom 96 Consumable Kit for Applied Biosystems TC (Sufficient for 5 x 96 rxn runs, Cat. No. 902803)

• Axiom 96 TC Plate Sealing Kit (Sufficient for 25 x 96 rxn runs, Cat. No. 902802)

Note: Beckman plates, reservoirs, and tips are not included in the kits prepared by Thermo Fisher Scientific. You must order all Beckman supplies directly from Beckman.

IMPORTANT! For troubleshooting and support purposes, we strongly recommend that you perform the gel QC and OD quantitation process controls after resuspension.

CAUTION! Never process samples in test mode. The assay will fail; all of your samples and reagents will be lost.

Figure 13 Prompt displayed when the custom run option, Run method in test mode, is selected.

IMPORTANT! The method preferences (Figure 12 on page 37) should be chosen prior to starting the method. These settings are not prompted for at runtime.

Table 13 Axiom 96 Consumable Kit for Biomek FXP (Sufficient for 4 x 96 rxn runs, Cat. No. 902800)

Labware item Part No. Quantity

Bio-Rad Hard-Shell 96-well Plate

203015 40

Eppendorf Deep Well Plate 203079 4


Chapter 3 Preparation before you startEquipment, consumables, labware, and reagents3

Table 14 Axiom 96 Consumable Kit for QC (Sufficient for 40 x 96 rxn runs, Cat. No. 902801)


OD Plate, UV 202609 40

Table 15 Axiom 96 Consumable Kit for off-deck Applied Biosystems TC (Sufficient for 5 x 96 rxn runs, Cat. No. 902803)


Bio-Rad Hard-Shell 96-well semi-skirted PCR Plate 203055 5

Costar Clear Polystyrene 96-Well Plates 202165 5

Table 16 Axiom 96 TC Plate Sealing Kit (Sufficient for 25 x 96 rxn runs, Cat. No. 902802)


Bio-Rad Metal Lid 202519 1

Bio-Rad Microseal P Pad 202958 1

Table 17 Guidance for consumable kit ordering

Consumable kit

Thermal cycler option

On-deck,

TRobot

Off-deck PTC-200

Off-deck Applied

Biosystems

Off-deck Eppendorf

Master Cycler pro S

Axiom™ 96 Consumable Kit for Biomek FXP (Cat. No. 902800)

Axiom™ 96 Consumable Kit for QC (Cat. No. 902801)

Axiom™ 96 Consumable Kit for Off-Deck Applied Biosystems TC (Cat. No. 902803)

Axiom™ 96 TC Plate Sealing Kit (Cat. No. 902802)



Labware and materials used on the Biomek workstation deck

Table 18 Labware and materials used on the Biomek workstation deck

Labware Supplier and Cat. No. Image

Biomek AP96 – P250 Pipette Tips (aqua box; pre-sterile, barrier)

Beckman CoulterCat. No. 717253

Biomek Span P250 Pipette Tips (green box; pre-sterile, barrier)


Biomek Span P1000 Pipette Tips (yellow box; pre-sterile, barrier, conductive)




Biomek Span P50 Pipette Tips (pink box; pre-sterile, barrier)

Beckman CoulterCat. No. A21586

Bio-Rad Hard-Shell 96-well Plate(available in multiple colors)

Part of the Axiom™ 96 Consumable Kit for Biomek FXP Cat. No. 902800

Plate Part No. 203015

Alternate Vendor:Bio-RadCat. No. HSP-9631

Eppendorf 96 Deep-well Plate, 2000 μL(available in multiple colors)

Part of the Axiom™ 96 Consumable Kit for Biomek FXP Cat. No. 902800


Alternate Vendor:Eppendorf951033481

OD Plate, UV Part of the Axiom™ 96 Consumable Kit for QCCat. No. 902801


Alternate Vendor:E & K ScientificEK-25801

Table 18 Labware and materials used on the Biomek workstation deck (Continued)




Lid, metal(arched, auto-sealing with P pads)

Part of the Axiom™ 96 TC Plate Sealing Kit Cat. No. 902802

Lid Part No. 202519P Pad Part No. 202958

Alternate Vendor:Bio-RadCat. No. MSL-2032 andP Pad Cat. No. MSP-1003

Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate • This consumable is required only if

using off-deck Applied Biosystems 2720 or Applied Biosystems 9700 Thermal cyclers

Part of the Axiom™ 96 Consumable Kit for Off-deck (Applied Biosystems) TCCat. No. 902803


Alternate Vendor:Bio-RadCat. No. HSS-9601

Plate, Costar Brand Serocluster round Bottom Plate from Corning• Note: this consumable is required

only if using an off-deck Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycle

Part of the Axiom™ 96 Consumable Kit for Off-deck (Applied Biosystems) TCCat. No. 902803


Alternate Vendors:VWR International Cat. No. 29442-392E&K Scientific Cat. No. EK 680568Corning Mfg Cat. No. 3795



Front view

Side view



The Applied Biosystems 9700 and the Applied Biosystems 2720 use the semi-skirted 96-well plates (Cat. No. HSS-9601). For use on the Biomek deck, the semi-skirted PCR plate must be stacked on a Costar brand Serocluster 96-well Round Bottom Microtitration plate as shown in the figure to the right.

Beckman Deep Well Titer Plate(polypropylene)


Frame for reservoirs Beckman CoulterCat. No. 372795

Half reservoirHalf module, 75 mL capacity




Frame



Quarter Reservoirs• Quarter module, 40 mL capacity• Quarter module divided by width,

19 mL capacity each receptacle

Beckman Coulter• Cat. No. 372790 (40 mL)• Cat. No. 372792 (19 mL)

Reagent block, chilled to 4°C Beckman CoulterCat. No. A83054

24-Position Tube Rackwith one 11 mm tube insert in position A6.

Beckman CoulterCat. No. 373661 (rack)Cat. No. 373696 (insert)



Divided by width19 mL capacity

Undivided40 mL capacity

Metal posts on block circled in red.A1

Tube insert A6



Adaptor, Deep Well Plate (installed on the Shaking Peltier)

This adaptor is typically installed by a Beckman Coulter field service technician during new system installation or a system upgrade. Ensure that you have one of these adaptors on the deck prior to running this assay.

Beckman CoulterCat. No. A83050

Reagent Block Template(designed specifically for use with the Axiom 2.0 Reagent Kit)

Contact Thermo Fisher Scientific

Zerostat Anti-static Gunand Ion-Indicator Cap

Milty Zerostat,

Thermo Fisher Scientific Cat. No. 74-0014



The metal block is the adaptor.

Template on reagent block. Metal posts on block circled in red.



GeneTitan MC Instrument ConsumablesAll consumables for the GeneTitan MC Instrument are provided by Thermo Fisher Scientific. The following table provides guidance on the consumables that are shipped with the array plate.

IMPORTANT! All covers must have barcodes. Discard any cover without a barcode.

Table 19 Axiom™ GeneTitan™ MC Instrument consumables (PharmacoScan™ 96F Array Plate)

Item Part number

Labware image Information

PharmacoScan 96F Array Plate

903160 PharmacoScan 96F Array Plate:• Comprised of three

parts: clear plastic cover, array plate, and blue array plate protective base.

• The clear plastic cover for the array plate protects the array plate during transport. Discard after opening pouch.

• The array plate must always be kept in the blue array plate protective base at all times.

• The blue array plate protective base in the package holds the array and protects it from damage or exposure to dust.

Note: Array plate is not included in the Axiom GeneTitan Consumables Kit.

Clear tray shipping cover (to be discarded)Array plate protective baseArray plate

1

2

3

1

2

3



Table 20 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606)

Item Part number Labware image Information

Scan Tray 900746 Box501006 Pouch

96-Plate Scan Tray:• Comprised of three

parts: scan tray, black protective base, and a scan tray cover.

• The black scan tray protective base in the package protects the glass bottom of the scan tray from damage before it is loaded into the GeneTitan MC Instrument.

• The scan tray cover protects the contents in the scan tray and must be deionized before used. See Appendix E, "Deionization procedure for GeneTitan™ trays and covers" on page 241.

• Remove the black scan tray protective base before loading the scan tray with the scan tray cover into the GeneTitan MC Instrument.

• The Scan Tray must be loaded into the GeneTitan Instrument with the Scan Tray Cover only.

• Do not load the Scan Tray with the protective base still on.



Black Scan Tray Protective Base, shown without the Scan Tray with cover

• The black scan tray protective base in the package is used to protect the bottom of the scan tray glass from damage. The black scan tray is distinct from the blue array plate protective base and must not be used with the array plate.

• Remove and set aside the protective base from the scan tray before loading.

Scan Tray with cover, shown without the black protective base

• The GeneTitan scan tray must be loaded with the scan tray cover into the GeneTitan MC Instrument.

• Do not load the scan tray with the protective base.

Table 20 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606) (Continued)




GeneTitan 5 Stain Trays Kit

4249910 Kit501025 Tray

• The GeneTitan Stain Tray Kit comes with 5 stain trays packaged in zip-top bags to keep them free of dust.

• The GeneTitan stain trays are barcoded and the trays have separator walls that are flush with the frame of the stain tray, as shown by the yellow line and the yellow oval in the lower photo.

GeneTitan™ Stain and Scan Tray Cover

202757 • The GeneTitan stain and scan tray covers prevent evaporation of the stains in stain trays and the array holding buffer in the scan tray.

• All stain and scan trays must be placed in the GeneTitan MC Instrument with the GeneTitan stain tray cover.

• All tray covers must be deionized to remove static electricity prior to placing the cover on the tray.

• See the section "Deionization procedure for GeneTitan™ trays and covers" on page 241 for the anti-static procedure.





Proper tray alignment and loading

Proper alignment and loading of plates, covers and trays is critical when using the GeneTitan MC Instrument. Each plate, cover and tray has one notched corner. The notched corner of plates, trays, covers and bases must be in vertical alignment with each other, and placed in position A1 per the Tray Alignment guide inside each GeneTitan MC Instrument drawer (Figure 14 and Figure 15 on page 53).

Note: Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading onto the GeneTitan MC Instrument.

Note: The instrument control software will display a warning if it detects a problem during the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and Rinse bottles should be replaced if the software displays such a warning. Refer to Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 247 for the

GeneTitan stain tray shown with the stain tray cover

Tray 501025Cover 202757

Hybridization Tray

900747 • After aliquoting the denatured hyb ready samples into the hybridization tray, the tray should be immediately loaded into the GeneTitan MC Instrument with the barcode facing away from the operator, i.e., Barcode should be on the back side.



IMPORTANT! When running a multi-plate workflow, you must pay careful attention to the software prompts that tell you which side of the drawer to place or remove a plate/tray.

CAUTION! Take care not to damage the consumables or bend the blue cover posts or scan tray posts.



message displayed to the user and the procedure for replacing the filters.

Figure 14 Proper alignment and loading of plates, covers and trays in the GeneTitan MC Instrument

Plates and trays must be seated in this rectangular recess.

The notched corner of all plates, bases, and covers and must be seated in this corner of the drawer per

Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading.

Notched corner of array plate aligned with notched corner of blue base.

IMPORTANT! Remove the plastic protective shipping tray cover. 1

2

3


1

2

344

5

5

6

7

7

6



Figure 15 Array plate with protective blue base and the hyb tray aligned and properly loaded into drawer 6

Array Plate with Protective Blue Base

Hyb Tray

1

2

1 2

IMPORTANT! When you install the consumables, ensure that the fingers are retracted. Do not lay the consumables on top of the drawer fingers - this indicates that the instrument is not functioning correctly. Please notify your Field Service Engineer if the fingers do not retract automatically. You should place the trays into the instrument drawers when a drawer is fully extended by the instrument. The fingers are retracted when the drawer is open and are extended when the drawer is closed in order to restrain the consumable. See Figure 16 for an image showing the location of the tabs.

Figure 16 Photo identifying the location of drawer tabs

2

1

Drawer tab, or “finger” in back.

Drawer tab, or “finger” on right side.

1

2



Stain trays and covers

Labeling GeneTitan hybridization and reagent traysWhen preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument, you will need to mark each tray in a way that identifies its contents.

Proper labeling for hyb trays and reagent trays is described in:

• ʺLabeling for hyb traysʺ, below

• ʺLabeling for stain traysʺ on page 56

Labeling for hyb trays

You may label the hyb tray on the front part of the short side of the tray, next to the notch at the left, as shown in Figure 18. The proper section for labeling is closest to the notched corner, corresponding to the A1 and B1 wells.

IMPORTANT! Always place the flat side of the cover against the stain tray.

Figure 17 Placement of covers on trays

Correct placement of cover on stain tray. Incorrect placement of cover on stain tray.

IMPORTANT! It is critical that you write only on the proper locations of the proper sides of hyb and stain trays. Do NOT write in any other location, as this can interfere with sensors inside the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.



Figure 18 Labeling GeneTitan hyb trays

CAUTION! Writing on the wrong side of the hyb tray may interfere with the operation of sensors in the GeneTitan MC Instrument.

Do NOT label trays on the long side of the tray.

Notched corner of the hyb tray should face the front.

Label the hyb tray in this area.

1

2

3

1 2 3



Labeling for stain trays

You may label the stain trays on the left side of the front of the tray as shown in Figure 19. The correct side is closest to the notched corner, corresponding to the A1 through C1 wells.

IMPORTANT! Do not confuse hyb trays with stain trays.

Figure 19 Labeling GeneTitan stain tray (stain tray shown with lid)


Notched corner of the stain tray should face the front.

Label the stain tray here.

1 2 3

1

2

3



Loading tray consumables onto the GeneTitan™ MC Instrument

Loading, or installing, the trays and plates onto the GeneTitan MC Instrument is a simple procedure, but there are certain precautions that you must take in order to ensure an error-free procedure. The following figures show you how to do it. See Figure 20 through Figure 23.

Figure 20 Scan tray loaded in drawer 2

IMPORTANT! When you load the plates, or trays, insert them under the tabs, or fingers, that may protrude into the stage. Confirm that the tray is not resting on these fingers. See Figure 63 on page 136.

Do NOT load the protective black base packaged with the scan tray.



Figure 21 Stain 1 tray and Ligation tray loaded in drawer 3

Figure 22 Stain 2 tray and Stabilization tray loaded in drawer 4



Reagent block template

The Axiom™ 2.0 Reagent Kit template fits precisely onto the top of the chilled reagent block, and is held in place by the metal posts on the block (Figure 24). Using this template will help ensure the proper placement of reagent tubes onto the block for each method.

Reservoir stickers

The reservoir labels are stick-on labels for the modular reagent reservoir frames for the different stages of the automated workflow. These stick-on labels are color-coded to match the colors found on the caps of the reagent tubes in the Axiom 2.0 Reagent Kit. Using these labels helps ensure the proper placement of reservoirs and reagents for each method.

Figure 23 Stain 1 tray loaded in drawer 5

Figure 24 Axiom 2.0 Reagent Template for the reagent block

CAUTION! The Reservoir Stickers for Windows® 7 (Part No. 101135) are different than the XP stickers. Please make sure you are using the correct version.



There are four reservoir holders used in the Axiom 2.0 method. Three of these will have templates on two sides and the remaining reservoir holder will have a template on one side for a total of 7 templates.

Remove the protective surface from the back of the label and place on the reservoir frames as directed in the Figure 25 through Figure 28.

Reservoir frame 1

Reservoir frame 2

Figure 25 Reservoir frame 1 for Windows® 7 users


Side A

Side B

Side A

Side B



Reservoir frame 3

Reservoir frame 4

Related Biomek FXP Target Prep Express documentation

The following user manuals are installed at the same time as the Biomek FXP Target Prep Express software (Start All Programs Beckman Coulter Manuals). Refer to these for troubleshooting the Biomek workstation.

• Biomek® Liquid Handler User’s Manual, Beckman Coulter Pub. No. 987834

• Biomek® Software User’s Manual, Beckman Coulter Pub. No. B30026AA



Side A

Side B

Side A


4 Multiplex PCR and Targetpreparation on the Biomek FXP

with Windows® 7

PharmacoScan™ Assay on the Biomek FXP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Stage 1A: Multiplex PCR (mPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Stage 1B: DNA amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Stage 2: Fragmentation and purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Stage 3: Centrifugation and drying pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Stage 4: Resuspension, Hybridization Preparation, and QC . . . . . . . . . . . . . . . . 96

Stage 5: Preparation for the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . 109

PharmacoScan™ Assay on the Biomek FXP

The PharmacoScan Assay 96-Array Format Automated Protocol leverages the Axiom 2.0 Assay method on the Biomek FXP for target preparation and GeneTitan reagent preparation to process 96 samples at a time. Please note that the multiplex PCR (mPCR) and mPCR spike-in steps are not part of the automated method and is executed off-deck, manually. The protocol is performed in two parts:

• Part 1: Target Preparation

– mPCR preparation and mPCR spike-in steps are performed manually, off deck.

– All other steps are performed on the Biomek FXP Target Prep Express

• Part 2: Array Processing is performed on the GeneTitan™ Multi-Channel (MC) Instrument

A list of all equipment and resources for the PharmacoScan Assay with Automated Target Prep is in the PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP Site Preparation Guide, Pub. No. 703473.

This chapter includes instructions for Part 1: Target Preparation.

Using the automated target preparation protocol, eight array plates can be processed per work week for a total of 768 arrays, 752 samples and 16 controls. See Chapter 6, ʺProcessing eight PharmacoScan™ array plates per weekʺ on page 171 for further information. There is also a protocol for processing three array plates per work week for a total of 288 arrays, 282 samples and 6 controls. See Chapter 7, ʺProcessing three PharmacoScan™ array plates per weekʺ on page 196 for further information.


Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 1A: Multiplex PCR (mPCR) 4

Stage 1A: Multiplex PCR (mPCR)

The following steps are necessary to perform mPCR:

ʺ1: Prepare for mPCRʺ on page 65

ʺ2: Prepare the mPCR master mixʺ on page 66

ʺ3: Set up the mPCR reaction plateʺ on page 66

ʺ4: Run the PharmacoScan mPCR thermal cycler protocol and proceedʺ on page 67

ʺ5: Store the mPCR reaction plateʺ on page 67

Duration For 96 samples:

• Time to thaw materials: 0.5 hr

• Hands-on time: approximately 0.5 hr

• Thermal Cycler run time: approximately 3.5 hr

• Total time required: approximately 4.5 hr

Input required The mPCR Sample Plate, with 10 µL of sample diluted to a concentration of 5 ng/µL in a 96-well PCR plate compatible with your thermal cycler.

See ʺGenomic DNA preparationʺ on page 17 for more information.

IMPORTANT! Before proceeding to mPCR or DNA Amplification, perform the gDNA preparation described in Chapter 2, ʺGenomic DNA preparation and requirementsʺ on page 14.

IMPORTANT! Pre-amplification preparation should take place in a dedicated area such as a biosafety hood with dedicated pipettes, tips, vortex, etc. See ʺPre-amplification areaʺ on page 15 for more information.

Figure 29 mPCR sample plate

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

C1

C2


Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 1A: Multiplex PCR (mPCR)4

Equipment, consumables and reagents required

Equipment and consumablesThe equipment and consumables listed in Table 21 are required for this stage.

Reagents required

Table 21 Equipment and consumables required for Stage 1A: mPCR

Quantity Consumable item

As required Adhesive seals for 96-well plate-Applied Biosystems MicroAmp clear adhesive film

1 Marker, fine point, permanent

1 96-well plate holder

1 15 ml conical tube (RNase/DNase-free)

Quantity Equipment

1 Vortex

1 Aluminum plate block cooled to 4°C

1 Plate centrifuge

1 Ice bucket with ice

1 Thermal cycler programmed with the PharmacoScan mPCR protocol (see "Thermal cycler recommendations and protocols" on page 25).

1 Each Rainin Pipettes: • Single channel P200• Single channel P1000• Multi-channel P200

As Needed Pipette tips

Table 22 Reagents Required for Stage 1A: mPCR

From the PharmacoScan Reagent Kit 96 Reactions Module

10X Primer Mix, 1 tube PharmacoScan Module A–20°C

Part No. 912896

User Supplied

From QIAGEN Multiplex PCR Plus Kit (100)1

(Cat. No. 206152)

• 2X Multiplex PCR Master Mix• 5X Q-Solution • RNase-free Water

1 CoralLoad Dye in kit is not needed for PharmacoScan Assay.

Quantity

3 tubes1 tube1 tube



1: Prepare for mPCR

To prepare for mPCR

1. Power on the thermal cycler programmed with PharmacoScan mPCR protocol (refer to Figure 5 on page 25). Make sure the heated lid option has been selected.

2. Thaw and prepare the mPCR Reagents and the mPCR Reaction Plate.

To thaw and prepare the reagents:

To thaw and prepare the mPCR Sample Plate

1. If the gDNA mPCR Sample Plate was frozen, thaw at room temperature on the benchtop, vortex, and pulse-spin.

2. Place mPCR Sample Plate on ice or a cold aluminum block once thawed.

To thaw and prepare the reagents from PharmacoScan Module A (Part No. 912896), PharmacoScan Reagent Kit

1. 10X mPCR primers

• Thaw on benchtop at room temperature then place on ice.

• Vortex and pulse-spin before use.

To thaw and prepare the reagents from QIAGEN Multiplex PCR Plus Kit (QIAGEN Cat. No. 206152)

1. QIAGEN 2x Multiplex PCR Master Mix:

• Obtain 3 tubes


• Invert each tube 10 times to thoroughly mix. Pulse-spin before use. DO NOT VORTEX.

2. Q-Solution, 5X

• Obtain 1 tube



3. 1 tube RNase-free Water

• Obtain 1 tube



IMPORTANT! gDNA samples must be 10 µL volume at a concentration of 5 ng/µL in a 96-well PCR plate (see Genomic DNA preparation, ʺmPCR sample plateʺ on page 20).

IMPORTANT! • Make sure reagents are thoroughly mixed prior to use.

• Vortex water and Q-Solution.

• Master Mix should be thoroughly mixed by inverting tube 10 times. DO NOT VORTEX.



2: Prepare the mPCR master mix

1. Label a 15 mL conical tube mPCR.

2. To the mPCR tube, add the reagents listed in Table 23 as follows:

a. Add water, Q-Solution, and primers to tube. Cap.

b. Vortex briefly. Pulse spin.

c. Add the QIAGEN 2X Master Mix to the tube. It will be necessary to use all three vials supplied by QIAGEN. It is recommended to set a P-1000 single-channel pipette to 800 uL and remove this volume from the first vial. Transfer solution to the 15-mL tube. Change tips, and repeat this step for the remaining two vials.

d. Mix thoroughly but gently, inverting tube end-over-end 10 times. Pulse spin.

3. The resulting mPCR Master Mix should be kept in ice and added to the mPCR sample plate as soon as possible after preparation.

3: Set up the mPCR reaction plate

1. Confirm the 96-well PCR sample plate is labeled, mPCR along with date and any desired experimental details.

2. Place the plate in an aluminum block which had been stored at 4°C.

3. Carefully pour the prepared mPCR Master Mix into a 25 mL reservoir.

4. Use a P-200 mutli-channel pipette to carefully transfer 30 µL of mPCR Master Mix into the mPCR plate. Final volume of each well is 40 µL.

5. Seal plate with adhesive seal, ensuring seal is firmly pressed down to prevent sample contamination during mixing and evaporation during PCR cycling.

6. Vortex plate for 2 sec in each quadrant twice (refer to ʺSeal, vortex, and spinʺ on page 28).

7. Spin down at 2000 rpm for 30 sec.

8. Return plate to cold aluminum block until plate can be loaded onto thermal cycler (Note: Load plate onto thermal cycler within five minutes).

9. Discard all leftover reagents, including any remaining unused QIAGEN reagents.

Table 23 mPCR Master Mix

Reagent 1 Reaction 120 Reactions

RNase-free Water 2 μL 240 μL

Q-solution 4 μL 480 μL

10X mPCR Primer Mix 4 μL 480 μL

2X QIAGEN Multiplex PCR Master Mix 20 μL 2400 μL

Total 30 μL 3600 μL



4: Run the PharmacoScan mPCR thermal cycler protocol and proceed

1. Place mPCR on thermal cycler and run PharmacoScan mPCR protocol (refer to Figure 5 on page 25).

Note: The mPCR Sample Plate is now referred to as the mPCR Reaction Plate from this point forward.

2. Proceed to ʺStage 1B: DNA amplificationʺ on page 69 after the mPCR Reaction Plate has been placed on the thermal cycler.

5: Store the mPCR reaction plate

1. After the PharmacoScan mPCR thermal cycler protocol is complete, remove the plate from thermal cycler, seal, and pulse-spin plate. Ensure plate is well sealed to prevent evaporation during storage.

2. Do one of the following:

• For mPCR spike-in and fragmentation on another day:

— Store mPCR Reaction Plate at –20°C.

• For mPCR spike-in and fragmentation later in the same day:

— If the mPCR spike-in and fragmentation will be carried out within 4 hours,store the plate in a refrigerator (2–8°C).

Note: An mPCR QC Gel can be run for qualitative evaluation of the mPCR reaction prior to the mPCR Spike-In step during Fragmentation. Refer to Appendix G, ʺmPCR quality control gel protocolʺ on page 257 for protocol details.



Figure 30 Stage 1A: mPCR Preparation workflow diagram

Stage 1A: mPCR Preparation

mPCR Preparation – Page 1 of 1

Final Volume: 40 μL/well

Q-solution

Prepare mPCR Master Mix – Part 1

Vortex briefly, Pulse-spin.

Labware and Reagents Needed

QTY 1 (QIAGEN Multiplex

PCR Plus Kit, Cat. No. 206152)

QTY 1

QTY 1QTY 1 (mPCR Sample Plate)

480 μLRNase-free Water

Run Multiplex PCRThermal Cycler Protocol

(Duration: ~3.5H)

30 μL/well

Prepare mPCR Master Mix – Part 22X QIAGEN Multiplex

PCR Master Mix

mPCR PlateSeal tight, Vortex,

Pulse-spin

Invert tube 10 times to mix, Pulse-spin

Pour into reservoir

2400 μL

10X mPCR Primer Mix

480 μL240 μL


Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 1B: DNA amplification 4

Stage 1B: DNA amplification

Note: For this protocol, the term samples includes the positive control.

Duration

Note: A 22-24 hr incubation is required at the end of this stage.

Equipment, consumables, labware and reagents required

Equipment and labware required

IMPORTANT! Before proceeding to DNA Amplification, do the gDNA preparation described in Chapter 2, ʺGenomic DNA preparation and requirementsʺ on page 14.

Table 24 Time required for "Stage 1B: DNA amplification"

Activity Time

Hands-on time ~30 min

Biomek FXP Target Prep Express ~19 min

Incubation 23 hr

Total time ~24 hr

Table 25 Equipment and labware required

Quantity Item


1 Benchtop cooler, chilled to –20°C

As required Kimwipes®


1 Mini microcentrifuge (microfuge with microtube rotor)

1 Oven (must maintain a constant temperature of 37°C for at least 23 hrs with a temperature accuracy of 1°C)• >3 array plates per week—we recommend using the BINDER ED 56• 3 array plates per week—OK to use the GeneChip Hybridization Oven

or the BINDER ED 56

1 Plate centrifuge

1 Vortex

Biomek workstation labware

1 box of each Barrier pipette tips:• Biomek Span P1000 (yellow)• Biomek AP96, P250 (aqua)

2 Plate, Bio-Rad Hard-Shell PCR 96-well


Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 1B: DNA amplification4

Reagents required

2 Plate, deep well titer

1 Reagent block, chilled to 4°C

3 Reservoir, quarter module (40 mL)

2 Reservoir, half module (75 mL)

Table 26 Reagents required for DNA Amplification

Axiom 2.0 Reagent Kit Module

Axiom 2.0 Denat Soln 10X

Module 1, –20°C

Axiom 2.0 Neutral Soln

Axiom 2.0 Amp Soln

Axiom 2.0 Amp Enzyme

Axiom Water

IMPORTANT! The Axiom 2.0 method used in the PharmacoScan Assay 96-Array Format Automated Protocol is compatible only with reagents from the PharmacoScan Reagent Kit 96 Reactions (Cat. No. 913025). These reagents are not interchangeable with reagents from other reagent kits, such as CytoScan™, SNP 6.0, DMET Plus, etc.

Table 25 Equipment and labware required (Continued)

Quantity Item



1. Perform the pre-run checklist

Check the water and waste containers

To check the system water and waste containers:

1. If necessary, fill the system water container using deionized water (no need for ultra-pure water).

2. If necessary, empty the system waste bottle.

Turn on the Biomek FXP Target Prep Express

To turn on the workstation:

1. Power on the workstation.

2. Ensure that all of the peripherals are powered on.

• Two Inheco Single Tec controllers

Each one controls the Static Peltier (Pelt_1) or the Shaking Peltier (SPelt_96); no additional power supply.

• Thermal cycler:

Biometra TRobot 96 if present on the Biomek workstation deck. Otherwise, a stand-alone thermal cycler can be used.

3. Launch the Biomek Software by double-clicking the Biomek Software icon on the desktop .

You can also open Start All Programs Beckman Coulter Biomek Software.

Home All AxesThis procedure will home all axes and prime the fluidics lines.

To Home All Axes:

1. Open Instrument Home All Axes.

2. Ensure all conditions in the Warning prompt Figure 31-A are met, then click OK.

An icon instructing you to Stop and wait while the instrument homes is displayed (Figure 31-B).

Figure 31 Homing All Axes

A B



3. When:

a. The Warning prompt in Figure 32-A is displayed, confirm that no tips are loaded in the Span-8 Pod, and click OK.

The lines for the Span-8 tips are primed and the next prompt shown in Figure 32-B is displayed.

b. When the intake (syringes and tubing) for the Span-8 tips is clear of bubbles, click OK.

2. Thaw and prepare the reagents and Sample Plate

Thaw and prepare the reagents and Sample Plate


1. Thaw the Sample Plate (containing 100 ng or 5 ng/µL of gDNA for human, 150 ng or 7.5 ng/µL of gDNA for diploid plants and animals, or 200 ng or 10 ng/µL of gDNA for polypoid plants and animals) on the benchtop at room temperature and spin.

Note: Do not place a frozen Sample Plate directly on the workstation deck.

2. Thaw the following reagents on the benchtop at room temperature.

• Axiom 2.0 Denat Soln 10X

• Axiom 2.0 Neutral Soln

• Axiom 2.0 Amp Soln

• Axiom Water

Leave the Axiom 2.0 Amp Enzyme in the freezer until ready to use.

Note: Allow ~1 hour for Axiom 2.0 Amp Soln to thaw on the benchtop at room temperature. If the solution is not completely thawed after 1 hour, vortex briefly and return to the benchtop to complete thawing. The bottles can also be thawed in a dish with Millipore water. The Axiom 2.0 Amp Soln must be thoroughly mixed before use.

3. Vortex all reagents (except Axiom 2.0 Amp Enzyme), then place at room temperature.

• Vortex the Axiom 2.0 Amp Soln and Axiom 2.0 Neutral Soln for 30 sec to thoroughly mix.

• Vortex and spin the Axiom 2.0 Denat Soln 10X before placing on the deck.

• For the Axiom 2.0 Amp Enzyme, just before placing on the deck gently flick the tube 3 times to mix and spin.

4. Preheat the Oven to 37°C.

We recommend using one of these ovens:

• BINDER ED 56

• GeneChip Hybridization Oven 645 (turn rotation on to 15 rpm)

Figure 32 Prompts displayed for priming the Span-8 pod fluidic lines

A B



3. Run the DNA Amplification step

To run the DNA Amplification step:

1. Open Project Open Project Axiom 2.0 Target Prep and click OK.

2. Open File Open to display the Open Method window.

Or click the Open Method icon .

3. Select Axiom 2.0 Target Prep and click OK

4. At the top of the main window (Figure 33-A), click the Run button to open the Axiom™ Target Prep window.

5. In the Axiom Target Preparation window (Figure 33-B):

a. Select DNA Amplification.

b. Click OK.

The Deck Layout for DNA Amplification is displayed (Figure 34 on page 75).

The Axiom 2.0 Target Prep project folder must be displayed in this menu.

1

1



6. Place the labware and reagents on the deck as directed in the following figures and table:

• Figure 34 on page 75—deck layout

• Figure 35 on page 76—location names of empty deck positions

• Table 27 on page 76—table detailing the labware, reagent and modular reservoir placement for the deck layout

• Figure 36 on page 77—reagent block

Figure 33 Opening the Axiom Target Preparation methods window

A B

IMPORTANT! Axiom 2.0 Amp Enzyme—Immediately prior to placing on the reagent block, gently flick the tube with your finger two to three times to mix; then spin. Do NOT vortex.



Figure 34 Deck layout window for the DNA Amplification method



Figure 35 Empty deck positions

Table 27 Labware and Reagent Locations on the Deck for the DNA Amplification Method

Position on Deck

Labware Reagent or Samples

TL1 Biomek AP96 – P250 Pipette Tips (aqua) —

P1 Beckman Deep Well Titer Plate gDNA samples

P4 Bio-Rad Hard-Shell 96-well plate (any color) —

P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)

Pour Axiom 2.0 Neutral Solution into Reservoir 2

P11 Reservoirs in frame:• Half module (1)• Half module (2)

• Pour Axiom Water into reservoir 1• Pour Axiom 2.0 Amp Soln into reservoir 2

Pelt_1 Reagent block, chilled to 4°C See Figure 36.

P14 Biomek Span P1000 Pipette Tips (yellow) —

P15 Bio-Rad Hard-Shell 96-well plate (any color)

SPelt96_1 Beckman Deep Well Titer Plate —

1Empty

Reservoir

2Axiom

2.0 Neutral

Soln

3Empty

Reservoir

1

Axiom Water

2

Axiom 2.0 Amp Soln



7. Check the deck layout to ensure that all labware and reagents are in the proper locations.

Note: If the physical deck does not match the Deck Layout and Confirmation window exactly (Figure 34 on page 75), either modify the physical deck to match exactly or choose Abort in the Deck Layout and Confirmation window.

8. Click OK.

The system flushes the Span-8 fluidics system. Observe the lines and syringes for air bubbles.

Figure 36 Placement of reagents on chilled reagent block for the DNA Amplification step.

Important:

• Position all plates and the chilled reagent block with A1 in the upper left corner of the frame.• Spin down all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.

Axiom 2.0Amp

Enzyme

Axiom 2.0 Denat Soln

10X

Reagent block with template.

Only the positions used for this method are shown.

Flick and spin the Amp Enzyme before placing in block.

Diagram of reagent block without template

A1

A1



9. At the prompt to repeat the Span-8 fluidics system flush (Figure 37):

• Click No if no air bubbles are present.

• Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.

The DNA Amplification step runs until the Amplification Master Mix has been added to the Sample Plate. Once complete, the instructions and prompt shown in Figure 38 are displayed.

• Follow the instructions for the Sample Plate (see also ʺUser interventionʺ below).

• Follow the instructions for clearing the workstation deck.

User intervention

1. Remove the Sample Plate.

2. Blot the top of the plate with a Kimwipe to remove any droplets that may be present.

3. Tightly seal the plate.

4. Vortex and pulse-spin the plate.

5. Place in a preheated oven and incubate at 37°C for 23 1 hr.

Note: If using a GeneChip Hybridization Oven, place the plate on the bottom of the oven. Plates do not rotate.

6. After 22 to 24 hr of incubation, do one of the following:

• Proceed directly to ʺStage 2: Fragmentation and purificationʺ on page 81

• Tightly seal and store the amplified samples at –20°C.

Figure 37 Flushing the Span-8 fluidics system to purge air bubbles

Figure 38 Instructions for clearing the workstation deck

IMPORTANT! • Seal the Sample Plate before placing in the oven.

• Always discard the used multi-channel pipette tips in position P3.

• Always store the reagent block at 4°C.



Summary of DNA Amplification

Incubate on Deck10 mins @ RT

Stage 1B: DNA Amplification

DNA Amplification – Page 1

Transfer Denat Soln 1X to Sample Plate

Prepare Denat Soln 1X & Distribution Plate

Span-8

Denat Soln 10X Axiom Water

488 μL 4392 μL

(4oC) (RT)D1 Reservoir

D1 Plate

30 μL/well

D1 Plate Sample Plate

20 μL/well

MC

Continued on AmplificationPage 2

Fill Neutral Soln Plate

N1 Reservoir N1 Plate

140 μL/well

[96] FORMAT TRANSFER MIX MOVE



Span-8MC

Continued from AmplificationPage 1

Prepare Amplification Master Mix & Distribution PlateAmp Soln Amp Enzyme

26374 μL 586 μL

(RT) (4oC)MM Reservoir

Amp Mastermix Plate

255 μL/well

DNA Amplification – Page 2

Stage 1B: DNA Amplification

Transfer Neutral Soln to Sample Plate

N1 Plate Sample Plate

130 μL/well

Incubate Sample PlateOffline for 23 (±1) hr. @ 37oC

Transfer Amplification Master Mix to Sample Plate

Amp Mastermix Plate Sample Plate

230 μL/well


Final Volume = 400 μL


Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and purification 4

Stage 2: Fragmentation and purification

Introduction To begin Stage 2, the mPCR Reaction Plate and DNA amplification must be complete. Before the DNA amplification Sample Plate is placed on the Biomek FXP deck for the Fragmentation step, you must spike-in 10 µL of PCR products from the mPCR Reaction Plate into the corresponding wells of the DNA amplification plate. After the mPCR spike-in, you are now ready to proceed with the Fragmentation step on the Biomek FXP.

Note: Purification is done by precipitation (See Step ʺ4. Precipitationʺ on page 89).

Duration

Input required • mPCR Reaction Plate from ʺStage 1A: Multiplex PCR (mPCR)ʺ on page 63.

• Sample Plate from ʺStage 1B: DNA amplificationʺ on page 69.



Table 28 Time required for "Stage 2: Fragmentation and purification"

Activity Time

Hands-on time ~25 min~50 min if frozen DNA from Step 1

Biomek FXP Target Prep Express– Deactivation incubation — 20 min to deactivate

the amplification reaction and 20 min to equilibrate to the fragmentation temperature

– Fragmentation incubation — 30 min

1 hr 20 min

Total time 1 hr 45 min to 2 hr 20 min

Table 29 Equipment, consumables and labware required

Quantity Item



1 Freezer, –20°C


As required Kimwipes



1 Oven, preheated to 37°C

1 Plate centrifuge

1 Vortex (for plates and microtubes)


Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and purification4

Reagents required


1 box of each Barrier pipette tips:• Biomek AP96, P250 (aqua)• Biomek Span P250 (green)• Biomek Span P1000 (yellow)

2 Plate, Bio-Rad Hard-Shell PCR 96-well

1 Eppendorf 96 Deep-well Plate, 2000 μL


3 Reservoir, quarter module

1 Reservoir, quarter module, divided by half

1 Reservoir, half module

Table 30 Reagents required for the Fragmentation Method

Reagent Module

From the PharmacoScan Reagent Kit 96 Reactions

Axiom Frag Enzyme (leave at –20°C until ready to use)Module 2-1, –20°C

Part No. 901528Axiom 10X Frag Buffer

Axiom Precip Soln 2

Axiom Frag DiluentModule 2-2, 2–8°C

Part No. 901529Axiom Frag Rxn Stop

Axiom Precip Soln 1

User-suppliedRefer to the PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP Site Preparation Guide, Pub. No. 703473

Isopropanol, 99.5% 96 samples: 65 mL

Table 29 Equipment, consumables and labware required (Continued)

Quantity Item




The following actions are the same as described under ʺ1. Perform the pre-run checklistʺ on page 71. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation.

To perform the pre-run checklist:

1. Power on the Biomek FXP Target Prep Express and all peripherals.

2. Check the water and waste containers; replenish or empty as required.


4. Home all axes (on page 71).

2. Thaw and prepare the amplified DNA samples and reagents

Thaw and prepare the Amplified DNA Sample Plate, mPCR Reaction Plate, and reagents

If the plate of amplified DNA Sample Plate is frozen:

1. Place the deep well plate in a small water bath.For example, pour Millipore water into a small tray. Place the frozen plate on the water in the tray.

2. Leave the plate in the water bath for ~50 min until all wells have thawed.

3. Spin down at 1000 rpm for 30 sec.

4. To avoid cross-contamination of wells during vortexing:

a. Remove the seal and blot the top of the plate with a Kimwipe.

b. Tightly reseal the plate with a fresh seal.

5. Vortex the plate for 30 sec to thoroughly mix.

6. Spin at 1000 rpm for 30 sec.

Thaw and prepare the mPCR Reaction Plate

If the mPCR Reaction Plate is frozen: (skip this step if the mPCR Reaction Plate was not frozen at the end of Stage 1A):

1. Thaw the plate at room temperature for about 30 minutes or until all samples are thawed.

2. Spin down the plate at 1000 rpm for 30 sec.

3. To avoid cross-contamination of wells during vortexing, remove the seal and tightly re-seal the plate using a fresh seal.



Thaw and prepare the reagents


1. Thaw the following reagents on the benchtop at room temperature.

• Axiom 10X Frag Buffer

• Axiom Precip Soln 2

2. Vortex all reagents (except Axiom Frag Enzyme), then place on ice.

• Vortex and spin Axiom Precip Soln 2 before placing onto deck.

• For the Axiom Frag Enzyme: Leave at –20°C until ready to use. Just before placing on the deck, gently flick the tube 3 times to mix and spin.



3: mPCR Spike-in to Amplification Plate

• If proceeding directly from the end of ʺStage 1B: DNA amplificationʺ on page 69, remove the amplified DNA Sample Plate from the 37°C oven.

• If working with a thawed amplified DNA Sample Plate, change the seal, vortex, and pulse-spin the Sample Plate.

1. Carefully transfer 10 µL of the mPCR reaction into the corresponding well of the amplified DNA Sample Plate. Pipet up and down a few times to ensure complete liquid transfer from pipette tip.

2. Seal plate well. Ensure the seal is securely attached to the plate to minimize evaporation during next steps.

3. Thoroughly mix by vortexing plate for 30 seconds and pulse spin.

4. Immediately proceed to the next step, Run the Fragmentation step.

4. Run the Fragmentation step

To open the Target Preparation methods window:


2. Click the Open Method icon, select Axiom 2.0 Target Prep, and click OK.

3. Click the Run button.

4. Select Fragmentation, then click OK (Figure 39).

The deck layout for Fragmentation is displayed (Figure 40).





• Figure 42 on page 87—reagent cold block

Figure 39 Selecting the Fragmentation method

IMPORTANT! Remove the seal from the Sample Plate before placing on the deck.



Figure 40 Deck layout for the Fragmentation method




Table 31 Labware and reagent locations on the deck for the Fragmentation step

Position on deck

Labware Reagent or samples

TL1 Biomek AP96 – P250 Pipette Tips (aqua) —

P5 Bio-Rad Hard-Shell 96-well plate —


P7 Beckman Deep Well Titer Plate Sample Plate(amplified gDNA with mPCR products)

P8 Eppendorf 96 Deep-well Plate, 2000 μL —

P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module, divided (3 and 4)

• Pour Axiom Frag Rxn Stop into reservoir 1• Pour Axiom 10X Frag Buffer into reservoir 2

P11 Reservoirs in frame:• Half module (1)• Quarter module (2)

• Pour Isopropanol into reservoir 1• Pour Axiom Precip Soln 1 into reservoir 2

Pelt_1 Reagent block, chilled to 4°C See Figure 42 on page 87.

P13 Biomek Span P250 Pipette Tips (green)

P14 Biomek Span P1000 Pipette Tips (yellow) —

3Empty

4Empty

1AxiomFragRxnStop

2Axiom10X Frag

Buffer

1

Isopropanol

2AxiomPrecipSol 1



6. Check the deck layout to ensure that the labware, reagents and samples are in the proper locations.

Note: If the physical deck does not match the Deck Layout and Confirmation window exactly (Figure 40 on page 85), either modify the physical deck to match exactly or choose Abort in the Deck Layout and Confirmation window.

7. Click OK to continue.

The system will automatically flush the Span-8 fluidics system. Observe the lines and syringes for air bubbles.

8. At the prompt to repeat the Span-8 fluidics system flush:



Figure 42 Placement of reagents on chilled reagent block for the Fragmentation step

IMPORTANT:

• Always position the chilled reagent block with A1 in the upper left corner of the frame.• Spin down all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.

Axiom Precip Soln 2

Axiom Frag

Diluent

Axiom Frag

Enzyme




Axiom Frag Enzyme:

Flick to mix 3X and spin immediately prior to placing on the chilled reagent block.

A1

A1



The Fragmentation step begins. The Sample Plate is incubated at 65°C to inactivate amplification.

9. If you selected Prompt for manual DNA quantitation in the default software settings, you will then be prompted to remove an aliquot of each sample for a optional quantitation process control (Figure 39). The plate will remain at 36.5°C until the aliquots have been collected. Remove 4 µL aliquots from each well into a 96 well PCR plate and set aside for later quantitation (e.g., using the Quant-iT™ PicoGreen® dsDNA Kit from Life Technologies). Immediately continue sample processing by placing the amplification reactions back onto the shaking peltier and clicking OK at the prompt shown in Figure 43.

If Prompt for manual DNA quantitation was not selected, the box in Figure 43 will not appear.

Note: Remain near the Biomek FXP Target Prep Express if you are going to remove aliquots for quantitation. Avoid leaving the samples at 36.5°C for a long period of time.

10. Once the Fragmentation Complete message box appears (Figure 44), follow the instructions in the message box discarding used labware and tips, storing unused tips and storing the cold block at 4°C. Click OK when ready to continue and proceed to Step ʺ4. Precipitationʺ on page 89.

Figure 43 Prompt for manual DNA quantitation

Figure 44 Fragmentation complete message



4. Precipitation To precipitate the DNA samples:

1. Remove the Precipitation Plate from the deck.

2. Blot the top of the plate with a Kimwipe and seal tightly.

3. Place the plate in a –20°C freezer overnight to precipitate.

4. Return to the Biomek workstation and clear the deck.



Summary of Fragmentation

mPCR Spike-In

mPCR Spike-In – Page 1 of 1

Final Volume: 410 μL/well

Proceed to Fragmentation

Addition of mPCR Products

mPCR + Amp PlateVortex, Pulse-spin

10 μL/well

Labware and Reagents Needed

QTY 1 Amp Plate

QTY 1 mPCR Plate

Amplification Plate



Fragmentation

Span-8MC

Fragmentation – Page 1

Inactivate DNA Amplification ReactionSample Plate

Incubate:

1) 20 min @ 65.0°C2) 20 min @ 36.5°C

SPelt

Continued on FragmentationPage 2


Prepare Fragmentation Master Mix & Distribution Plate

Frag Diluent Frag Enzyme

1551.2 μL 150.6 μL

(4oC) (4oC)Frag MM Reservoir

Frag MM Distribution Plate

67 μL/well

FragB Reservoir

(RT)

6882.4 μL



Prepare Precipitation Soln & Distribution Plate

Span-8MC

Continued from FragmentationPage 1

Incubate on Deck

30 mins @ 36.5oC

Transfer Fragmentation Master Mix to Sample Plate

Sample Plate

57 μL/well

Fill Stop Solution Plate

Stop Reservoir Stop Plate

29 μL/well

Precip Soln 2 Precip Soln 1

222 μL

(4oC)

Precip Plate

240 μL/well

Continued on FragmentationPage 3


Fragmentation


FragMM Distribution Plate

Transfer Isopropanol to Precipitation Plate

Iso Reservoir Precip Plate

600 μL/well



Fragmentation

Span-8MC


Continued from FragmentationPage 2

Transfer Stop Solution to Sample Plate

Stop Plate Sample Plate

19 μL/well

Offline Precipitationsee user guide

Transfer Sample to Precip Plate

Sample Plate Precip Plate

Plate Contents




Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 3: Centrifugation and drying pellets4

Stage 3: Centrifugation and drying pellets

Duration

Equipment and consumables required

To centrifuge and dry the pellets:

1. Turn the oven on and preheat to 37°C.

Use an oven that can sustain a constant temperature of 37°C and has a temperature accuracy of 1°C (we recommend the BINDER ED 56). If processing three or fewer array plates, you can use a GeneChip Hybridization Oven.

2. Transfer the Precipitation Plate from the –20°C freezer to a pre-chilled centrifuge. Centrifuge the plate for 40 min at 4°C at 3200 xg (4000 RPM for the Eppendorf 5810R centrifuge with the rotor configuration described in the PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP Site Preparation Guide, Pub. No. 703473).

Table 32 Time required for Centrifugation and drying pellets

Activity Time

Hands-on time 10 min

Centrifugation 40 min

Drying 25 min

Total time 75 min

Table 33 Equipment and consumables required

Quantity Item


As required Kimwipes

1 Plate centrifuge, precooled to 4°C

1 Oven, preheated to 37°C

Sample Plate One plate of precipitated samples from Stage 2 in an Eppendorf 96 Deep-well Plate

CAUTION! During this step, handle the plate gently to avoid disturbing the pellets. Do not bump or bang the plate.

WARNING! Use rotor buckets with a soft rubber bottom to ensure that the deep well plates do not crack. Do not use buckets where the plates sit directly on a metal or hard plastic bottom, such as the A-4-62 rotor with a WO-15 plate carrier (hard bottom) for the Eppendorf 5810R centrifuge. Use of hard bottom plate carriers may result in cracked plates, loss of sample, unbalanced centrifugation, damage to the instrument and possible physical injury.


Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 3: Centrifugation and drying pellets 4

3. Immediately after the 40 min centrifugation period, empty the liquid from the plate as follows:

a. Remove the seal.

b. Invert the plate over a waste container and allow the liquid to drain.

c. While still inverted, gently press the plate on a pile of Kimwipes on a bench and leave it for 5 min.

4. Turn the plate right side up and place in an oven for 20 min at 37°C to dry.

Note: If using a GeneChip Hybridization Oven, place the plate on the bottom of the oven. Plates do not rotate.


• Proceed directly to ʺStage 4: Resuspension, Hybridization Preparation, and QCʺ on page 96, even if some droplets of liquid remain. Leave the Sample Plate at room temperature. It is helpful to begin preparing reagents for Stage 4 while centrifuging and drying pellets.

• Store the plates for resuspension later in the same day:

• Tightly seal the plates.

• If resuspension will be carried within 4 hours, keep the plates at room temperature.

• If resuspension will be carried out in more than 4 hours, store the plates in a refrigerator (2–8°C).

• Store the plates for resuspension on another day:

• Tightly seal the plate and store at –20°C.


Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC4

Stage 4: Resuspension, Hybridization Preparation, and QC

Note: In this stage, resuspension buffer is delivered to the plate of pelleted DNA by the Biomek FXP Target Prep Express. The samples are then resuspended by shaking off deck. The user must pay careful attention to pop-up prompts that inform the user when to remove the plate from the deck to perform an off-deck pellet resuspension step, and when the user must replace the plate with resuspended DNA back on the deck to complete the method.

Duration Resuspension and hybridization preparation



Table 34 Time required for Resuspension

Activity Time

Hands-on time 15 min

Frozen pellet equilibration to room temperature 1.5 hr

Biomek FXP Target Prep Express 31 min

Table 35 Equipment, consumables and labware required

Quantity Item






1 Plate centrifuge, at Room Temp

1 Vortex


1 box of each Barrier pipette tips:• Biomek Span P50 (pink)• Biomek AP96, P250 (aqua)• Biomek Span P250 (green)• Biomek Span P1000 (yellow)


Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC 4

Reagents required

5 platesOR:

Bio-Rad Hard-Shell PCR 96-well (HSP-9631)For on-deck thermal cycling or when using the PTC-0240/PTC-200 off-deck thermal cycler

6 plates for off deck

cycling with Applied

Biosystems thermal cyclers

4 Bio-Rad (HSP-9631) Hard-Shell PCR 96-well Plate and 1 HSS-9601 Hard-Shell Full-Height 96-Well Semi- Skirted PCR Plate1 Plate, Costar Brand Serocluster round bottom plate

For off-deck thermal cycling using the Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycler– Note: The HSS-9601 plate stacked on the Costar brand serocluster

round-bottom plate should only be used on position P2 on the Biomek FXP deck. See Figure 52 on page 112.

1 Plate, OD


1 Reservoir, 19 mL (quarter module, divided)

3 Reservoir, 40 mL (quarter module)

Table 36 Reagents required for Resuspension and Hybridization

Reagent Module

From the Axiom 2.0 Reagent Kit

Axiom Hyb Buffer Module 2-1, –20°C

Axiom Hyb Soln 1

Axiom Resusp Buffer Module 2-2, 2–8°C

Axiom Hyb Soln 2

Other reagents required

Nuclease-Free Water, ultrapure MB grade(Thermo Fisher Scientific, Cat. No. 71786; for OD and gel plate preparation)

To fill line of divided reservoir

TrackIt Gel Loading Buffer, diluted 1000-fold(see Appendix B, "Fragmentation quality control gel protocol" on page 226 for dilution instructions.)

13 mL

Table 35 Equipment, consumables and labware required (Continued)

Quantity Item



1. Preparing frozen pellets and Axiom Resusp Buffer

The equilibration of resuspension buffer and pellets to room temperature (18°C to 25°C) is very critical for the success of the Axiom 2.0 assay. When either is cooler than room temperature, pellets may not resuspend completely. This may result in compromised assay performance. Please note following guidelines on how to work with plates with fresh, cold or frozen pellets:

Pellets:

• Plates with fresh pellets can be kept at room temperature for up to 4 hours if proceeding with the resuspension and hybridization protocol within an hour of this time frame.

• Plates with fresh pellets that are not processed within 4 hours can be transferred to a refrigerator (2-8°C) for few hours only if processed within a day. However, it is critical to equilibrate the plate to room temperature for at least 30 minutes before proceeding with the resuspension and hybridization protocol.

• Plates with frozen pellets (e.g., on Day-5 of 8-plate workflow) must be pre-equilibrated at room temperature for at least 1.5 hour before proceeding with the resuspension and hybridization protocol.

Resuspension Buffer:

• Resuspension buffer, when pulled out from Module 2-2 at 2-8°C, needs at least 1 hour to equilibrate to room temperature.






3. Launch the Biomek software.


3. Thaw and prepare the reagents

Thaw and prepare the reagents


1. Thaw Axiom Hyb Soln 1 and warm Axiom Resusp Buffer on the benchtop at room temperature.

2. Vortex the Axiom Resusp Buffer and the Axiom Hyb Buffer.

3. Vortex and spin Axiom Hyb Soln 1 and Axiom Hyb Soln 2.

4. Run the Resuspension, Hybridization Preparation, and QC step

Note: We strongly recommend that you run two quality process controls during this step:

• A gel to verify successful fragmentation

• An OD quantitation of each resuspended sample

The Biomek FXP Target Prep Express can be set to prepare fragmentation and OD plates that are ready for processing. These process controls must be selected as a run preference prior to starting a run. See ʺSetting method preferencesʺ on page 35 for instructions.



To run the Resuspension, Hybridization Preparation, and QC step:



3. Click the Run button.The Axiom 2.0 Method selection dialog box appears.

4. Select Resuspension, Hybridization Preparation, and QC and click OK.The deck layout for this method is displayed (Figure 45 on page 100).






6. Label the Bio-Rad plates placed on the deck in positions P2, P9 and P11. For example:

• P2—Hyb Ready + <sample description>

• P9—Dil QC

• P11—Gel QC

Note: Verify the appropriate plastic consumables are being used on the deck for the Hyb Reaction Plate when using the Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycle.

IMPORTANT! The pellets and the resuspension buffer must be at room temperature before proceeding with this step.



Figure 45 Deck layout for the Resuspension, Hybridization Preparation, and QC

If Prepare plates for gel QC and OD after hyb rxn transfer is selected in method preferences, the following labware is required on the deck:

• Multichannel P50 pipette tips

• Dilution QC plate• Gel QC plate• OD QC plate (do NOT touch

bottom of plate)If not selected, these labware are not needed and will not appear in the deck layout.

See "Setting method preferences" on page 35 for more information.




Table 37 Labware and reagent locations on the deck for Resuspension, Hybridization Preparation, and QC

Positionon deck


TL1 Biomek AP96 – P250 pipette tips (aqua) —

P11 Biomek Span P50 Pipette Tip (pink) —

P2 Hyb Reaction Plate:• On-deck TRobot 96 or off-deck PTC-200 or PTC 0240 users:

Bio-Rad Hard-Shell 96-well plate (HSP-9631) or

• Off-deck Applied Biosystems 9700 or 2720 users: Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (HSS-9601) stacked on a Costar Round Bottom plate

WARNING: When using the Applied Biosystems 9700 or the Applied Biosystems 2720 off-deck thermal cycler for denaturing the Hyb Ready Plate, the Bio-Rad HSS-9601 Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate must be stacked on a CoStar Brand Serocluster Round Bottom Plate (Axiom 96 Consumables kit for off-deck Applied Biosystems users (Part No. 902803)) as shown in Table 18 on page 44.The HSS-9601 and HSP-9631 PCR plates are not interchangeable on the Biomek FXP deck.

P3 Eppendorf 96 Deep-well Plate, 2000 μL Pelleted samples


P8 Bio-Rad Hard-Shell 96-well plate

P91 Bio-Rad Hard-Shell 96-well plate Dilution QC Plate

P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)• Quarter module, divided (4 and 5)

• Pour Axiom Resus Buffer into reservoir 1• Pour Axiom Hyb Buffer into reservoir 2• Leave reservoir 3 empty• Pour Gel Diluent (diluted gel loading buffer) into reservoir 4:

13 mL• Pour nuclease-free water into reservoir 5 to fill line

1Axiom

ResuspBuffer

2AxiomHyb

Buffer

3Empty

Reservoir

4Gel

Diluent

5Water



P111 Bio-Rad Hard-Shell 96-well plate (for Gel QC) Gel QC Plate

P121 OD plate, 96-well UV OD QC Plate

Pelt_1 Reagent block, chilled to 4°C See Figure 47 on page 102.

P13 Biomek Span P250 pipette tips (green) —

P14 Biomek Span P1000 pipette tips (yellow) —

1 If QC is not selected, leave deck positions P1, P9, P11, and P12 empty.

Table 37 Labware and reagent locations on the deck for Resuspension, Hybridization Preparation, and QC

Positionon deck


Figure 47 Placement of reagents on chilled reagent block for Resuspension, Hybridization Preparation, and QC

IMPORTANT:


Axiom Hyb

Soln 2

Axiom Hyb

Soln 1




A1

A1




Note: If the physical deck does not match the Deck Layout window exactly (Figure 45 on page 100), either modify the physical deck to match exactly or choose Abort in the Deck Layout window.

8. Click OK to continue the method.





10. There are two pop-up prompts that inform the user: 1) when to remove the plate from the deck to perform an off-deck pellet resuspension step, and 2) when the user must return the plate with resuspended DNA back on the deck to complete the method.

Note: Seal the resuspension plate tightly before placing it on the off-deck microplate shaker.

a. The first prompt appears following the addition of the Resuspension Buffer to the DNA pellets (see Figure 48).

• Remove the Sample Plate from Position 3. This plate contains the DNA pellets in Axiom Resuspension Buffer.

b. Follow the instructions below to carry out the DNA pellet resuspension by off-deck shaking. Upon completion of the on-deck method that aliquots the Axiom Resuspension Buffer to the Sample Plate containing the pelleted samples, resuspension is carried out by shaking off-deck using the following steps:

• Blot the top of the Sample Plate using a Kimwipe to remove any droplets that may be present.

• Seal the plate tightly; blue pellets should be visible at the bottom of the wells.

• Place the Sample Plate onto one of the following shakers for 10 minutes:

– Thermo Scientific Compact Digital Microplate Shaker: set at 900 rpm

– Jitterbug: set at speed of 7

– Inspect the plate from the bottom. If the pellets are not dissolved, repeat the shaking step.

– Pulse-spin the plate in a room temperature centrifuge.

c. Ensure that the instructions in the pop-up message box were followed.

d. When ready, click OK to proceed with the method.

The method continues by preparing the Hybridization Master Mix and, if selected, the Sample QC Plates.

Figure 48 Instructions to perform the off-deck DNA pellet resuspension.



e. A second prompt appears which guides the user to return the Sample Plate back on the deck to prepare the final Hyb Reaction Plate.

f. Pulse-spin the Precipitation Plate with resupended DNA samples to get all the droplets down, minimizing sample loss.

g. Unseal the Precipitation Plate before placing it back on the deck at Position 3.

h. Ensure that the instructions in the pop-up message box were followed.

i. When ready, click OK to proceed with the method.

The method continues by preparing the Hyb Reaction Plate (the final Hyb Ready samples) and, if selected, the Sample QC Plates.

11. Run the fragmentation gel and OD quantitation process controls.

For instructions and guidelines on assessing gel and OD results, see:

• Appendix B, ʺFragmentation quality control gel protocolʺ on page 226

• Appendix C, ʺSample quantitation after resuspensionʺ on page 229


• If the GeneTitan MC Instrument is free, and if the gel and OD quantitation results are good:

• Discard used labware and reagents

• Discard used tips

• Store unused Span-8 tips

• Click OK in the Resuspension and Hyb Prep complete dialog box and proceed to ʺStage 5: Preparation for the GeneTitan™ MC Instrumentʺ on page 109.

• If the GeneTitan MC Instrument is not free, then follow the instructions shown in Figure 49:

• Tightly seal the Hyb Rxn Plate and store at –20°C.

• Discard used labware and reagents

• Discard used tips

• Store unused Span-8 tips

• Store cold block at 4°C.

• Click OK when complete.

IMPORTANT! Only proceed with the method once the DNA pellets have been fully resuspended into Axiom Resuspension Buffer using an off-deck microplate shaker.

Figure 49 Instructions to follow if the GeneTitan MC Instrument is not free.



Summary of Resuspension and Hybridization Preparation

Resuspension & Hybridization Prep

Span-8MC

Resuspension & Hybridization Prep – Page 1

Prepare Resuspension Buffer Plate

ResB Reservoir ResB Plate

45 μL/well

Transfer Resuspension Buffer to Precipitation Plate

ResB Plate Precip Plate

35 μL/well

Prepare Hybridization Master Mix & Distribution Plate

Hyb Soln 2 Hyb Soln 1

1197 μL 66.5 μL

(4oC) (4oC)MM Reservoir

Hyb MM Plate

90 μL/well

y

Hyb Buffer

(RT)

9376.5 μL

Continued on ResuspensionPage 2


Follow User-Interface instructions to perform the

Off-line Pellet Resuspension(see user guide for details)

You must click “OK” to continue with method

A second message will appear later when it is time to place the Sample Plate with

resuspended DNA back on deck.

Assay




Resuspension & Hybridization Prep

Span-8MC

Resuspension & Hybridization Prep – Page 2

Continued from ResuspensionPage 1

Transfer Hybridization Master Mix to Precipitation Plate

Hyb MM Plate Precip Plate

80 μL/well

See QC flowchart if performing analysis after Resuspension

Transfer Hybridization Mix to Hyb Reaction PlatePrecip Plate Hyb Rxn Plate

115 μL/well

Store HybridizationReaction Plate



Follow User-Interface instructionsto return the sample plate back on the deck

after completing the Off-line Pellet Resuspension(see user guide for details)

Click “OK” to continue with method



QC

Span-8MC

QC – Page 1

Fill Dilution QC Plate with Water

Water Reservoir Dilution QC Plate

55 μL/well

Fill OD QC Plate with Water

Water Reservoir OD QC Plate

90 μL/well

Fill Gel QC Plate with Dye

Dye Reservoir Gel QC Plate

120 μL/well

Continued on QCPage 2

Transfer Hybridization Mix to Dilution PlateHyb Rxn Plate Dilution QC Plate

5 μL/well



Wait for the message prompt on whento return the sample plate back on the deck

after completing the Off-line Pellet Resuspension(see user guide for details)

Follow the instructions on the pop-up message and Click “OK” to continue with method



Perform Offline Analysis

QC

Span-8MC

QC – Page 2

Transfer Diluted Sample to OD QC PlateDilution QC Plate OD QC Plate

10 μL/well

Continued from QCPage 1

Transfer Diluted Sample to Gel QC PlateDilution QC Plate Gel QC Plate

3 μL/well





Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4

Stage 5: Preparation for the GeneTitan™ MC Instrument

About Stage 5 You will proceed to Stage 5 in one of two ways:

• Directly from Stage 4 without interruption.

• With samples that were stored at –20°C after Stage 4.

This stage, Preparation for GeneTitan, can include any combination of the options shown in Figure 50. The first two options complete target preparation on the Biomek FXP Target Prep Express.

The options are:

Option 1

• Denature samples - the Hyb Rxn plate is placed on the thermal cycler and the samples are denatured. At this step, you must also select the “Transfer denatured samples to hyb tray” option. After the denature program has completed, the block will hold temperature until the user has dismissed the prompt, indicating that they are ready to continue the method to transfer the samples to the hyb tray and then carry it to the GeneTitan MC Instrument. Do not leave samples on the thermal cycler for a long period of time.

Option 2

• Transfer denatured samples to hyb tray - the denatured samples are transferred from the Hyb Rxn plate to the hyb tray, and are ready to load onto the GeneTitan MC Instrument.

Note: When using an Applied Biosystems 9700 or the Applied Biosystems 2720 thermal cycler for off-deck denaturation, the Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (Hyb Reaction Plate) must be stacked on a Costar brand Serocluster Round Bottom plate from Corning (Corning Mfg Cat. No. 3795)

Figure 50 Software setup for Preparation for GeneTitan

You can run one step, or a combination of steps.


Chapter 4 Multiplex PCR and Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4

Option 3

• Prepare GeneTitan™ reagent trays - the solutions required for the fluidics stage of array processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays (three stain trays, one ligation tray, one stabilization tray and one scan tray with Holding Buffer).

When performing a 1 plate workflow, select two options (option 1 and option 2).

• Denature samples and Transfer denatured samples to hyb tray when you are preparing the samples to begin hybridization in the GeneTitan MC Instrument.

- or select one option (option 3)

• Prepare GeneTitan™ reagent trays to prepare reagents for loading onto the GeneTitan MC Instrument the following day when the plate is finished with the hybridization period and about to begin GeneTitan fluidics processing.

Options for performing a multi-plate workflow

When performing a multi-plate workflow (see Chapter 6, ʺProcessing eight PharmacoScan™ array plates per weekʺ on page 171 for a description of the 8 plate workflow), the need to carry out preparation of reagents for one plate while simultaneously hybridizing a second plate will arise. In this case, all three options in the Biomek FXP method can be carried out concurrently. Select all three options when using an on-deck thermal cycler.

• Denature samples and Transfer denatured samples to hyb tray will prepare samples for the new plate going into the GeneTitan MC Instrument to begin hybridization.

-and-

• Prepare GeneTitan™ reagent trays will prepare reagents for the plate that is already in the GeneTitan MC Instrument hybridization oven and is ready to go into the Wash, Ligation, Stain, and Scan phases of GeneTitan array processing.

Note: In the 8 plate workflow, all three options will only be selected for middle days of the workflow. On the first day of the workflow, only the Denature samples and Transfer denatured samples to hyb tray options will be used. On the last day of the workflow, only the Prepare GeneTitan™ reagent trays option will be used.

Note: When using an off-deck thermal cycler, avoid letting the samples sit at room temperature for an extended period of time after denaturation. Do not begin denaturation at the same time as the GeneTitan reagent preparation.

Off-deck thermal cycler option

The steps for performing a simultaneous preparation of GeneTitan reagent trays and hybridization of a second plate required in the course of a multi-plate workflow are modified slightly when the off-deck thermal cycler option is selected. The reagent trays to be loaded into the GeneTitan MC instrument are prepared in an initial run of the method.

Denaturation of the hyb ready samples in the off-deck thermal cycler begins mid way through the GeneTitan reagent prep on the Biomek deck.

After loading the reagent trays into the GeneTitan MC instrument, you must perform a second run of the Biomek method to transfer the denatured samples to the hyb tray for loading into the GeneTitan MC instrument. You will need a timer for this modified step.



The modified steps are:

1. Select Preparation for GeneTitan step with only the Prepare GeneTitan™ reagent trays box checked, click OK.

2. Prepare deck as shown in Figure 51.

3. Click OK.

4. Start timer. Wait 25-30 minutes, then begin denaturation of the hyb ready samples using the off-deck thermal cycler.

5. Upon completion of the GeneTitan reagent tray preparation, load reagent trays and scan tray into the GeneTitan MC instrument

6. Once the reagents are loaded into the GeneTitan MC instrument and the denaturation method on the thermal cycler is complete, retrieve the denatured hyb ready samples from the thermal cycler

7. Return to Biomek FXP and begin a new method, select Preparation for GeneTitan step with only the Transfer denatured samples to hyb tray box checked, click OK.

8. Prepare deck as shown in Figure 52 (note that a spacer must be used with HSS-9601 plates for Applied Biosystems thermal cyclers).

Figure 51 Deck layout for “Prepare GeneTitan™ Reagent Trays” only (selected for Off-deck Thermal Cycler option)



9. Click OK.

10. After the denatured samples have been transferred to the GeneTitan hyb tray, load hyb tray into the GeneTitan MC Instrument.

Figure 52 Deck layout for “Transfer Denatured Samples to Hyb Tray”: Off-deck Thermal Cycler option and position P2

-

Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted

PCR Plate (HSS-9601) stacked on a Costar Round

Bottom plate for off-deck thermal cycling with the

Applied Biosystems 9700 or 2720 thermal cycler

The Hyb Reaction Plate in deck position P2 must be

one of the following:

Bio-Rad Hard-Shell 96-well plate (HSP-9631) for

on-deck or off-deck thermal cycling using the

TRobot 96, PTC-200 or PTC 0240

OR

--



If a plate is already in the hybridization ovenFor the plate that is already in the GeneTitan hybridization oven and that is ready to go into Ligation, Wash-Stain and Scan, select option 3, Prepare GeneTitan™ reagent trays. The solutions required for the fluidics stage of array processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays (three stain trays, one ligation tray, one stabilization tray and one scan tray with Holding Buffer).

Duration of GeneTitan™ MC Instrument reagent preparation and sample preparation

Sample denaturation

Denatured sample transfer to hyb tray

IMPORTANT! The reagent trays prepared in the third sub-step, Prepare GeneTitan™ reagent trays:

• Are NOT for use with the hyb tray currently being prepared on the Biomek workstation.

• Are for the continued processing of an Axiom array plate that is:

– already on the GeneTitan MC Instrument.

– has completed the hybridization stage.

– is ready for transfer to the fluidics area.

The reagent trays for the fluidics stage on the GeneTitan MC Instrument CANNOT be prepared in advance. Do not prepare these plates if there is no array plate ready for the fluidics stage. Once prepared, these plates must be loaded onto the instrument as soon as possible and cannot be stored.

Table 38 Time required to denature samples

Activity Time

Hands-on time: ~3 min

Biomek FXP Target Prep Express 17 min

Total time ~20 min

Table 39 Time required to transfer denatured samples to the hyb tray

Activity Time

Hands-on time 2 min


Total time ~4 min



Preparation of GeneTitan Reagent Trays

Note: When you select Denaturation and Preparation of the GeneTitan Reagent Trays, the on-deck processes run concurrently.

Equipment and consumables required

Sample Denaturation

Transfer to hyb tray

Table 40 Time Required to Prepare Reagent Trays for the GeneTitan MC Instrument

Activity Time

Prepare reagents (thaw and organize reagents)

~30 min

Hands-on time ~15 min


Total time ~75 min

Table 41 Equipment required for denaturing sample

If denaturing samples: Item Quantity

• On the Biomek workstation Lid, arched metal 1

Table 42 Consumables required for transferring denatured samples to a hyb tray

Item Quantity

Item required from the GeneTitan™ Consumable Kit, Cat. No. 901606:• hyb tray 1

Pipette tips, Biomek AP96 – P250 (aqua) 1 full box

If using off deck Applied Biosystems 9700 or Applied Biosystems 2720 thermal cyclers: Costar brand Serocluster Round Bottom plate from Corning Mfg. Cat. No. 3795) to be used for stacking under the HSS-9601 semi-skirted plate (Hyb Reaction Plate).

1



Prepare GeneTitan™ reagent trays

Note: See Table 19 on page 47 for GeneTitan MC Instrument Consumable catalog numbers.








Table 43 Consumables and other equipment required

Item Quantity

Items required from the GeneTitan™ Consumable Kit, Cat. No. 901606: • Scan tray with cover and protective base• Stain trays• Covers for stain trays

155

Pipette tips, Biomek AP96 – P250 (aqua) 1 full box

Pipette tips, Biomek Span P1000 (yellow) 1 full box

Pipette tips, Biomek Span P250 (green) 1 full box

Reagent block, chilled to 4°C 1

Reservoirs, quarter module divided 3

Reservoirs, quarter module 3

Tube rack, 24 position with insert (room temperature rack) 1

Zerostat Anti-Static Gun 1



2. Prepare the reagents for GeneTitan Reagent Tray Preparation

Note: Ligation Buffer and Ligation Solution 2 require approximately 30 to 40 min to thaw on the benchtop at room temperature.

Reagents required

Preparing Axiom Wash A and Axiom Stabilize DiluentDuring storage of the Axiom Wash A and Axiom Stabilize Diluent (in Module 4-2 stored at 4°C), precipitation in the form of clear crystals can sometimes occur. Therefore, follow the procedure below to ensure that any precipitate is returned to solution prior to use.

Note: The presence of some precipitate is okay and will not adversely impact assay performance. Follow the instructions below to resuspend any precipitate before use.

Table 44 Reagents required for GeneTitan MC Instrument Reagent Tray Preparation

Module Reagent Thaw on benchtop, then place on ice

Place on ice Place on benchtop at room temperature

Module 3Room

Temperature

Axiom Wash Buffer A

Axiom Wash Buffer B

Axiom Water

Module 4-1–20°C

Axiom Ligate Buffer - for 30 min

Axiom Ligate Enzyme Keep at –20°C until ready to use

Axiom Ligate Soln 1

Axiom Probe Mix 1

Axiom Stain Buffer

Axiom Stabilize Soln

Module 4-22 to 8°C

Axiom Ligate Soln 2 - for 30 to 40 min

Axiom Probe Mix 21

Axiom Wash A - for 30 min

Axiom Stain 1-A1

Axiom Stain 1-B1

Axiom Stain 2-A1

Axiom Stain 2-B1

Axiom Stabilize Diluent

Axiom Water

Axiom Hold Buffer1

1 These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time.



To prepare the Axiom Wash A:

1. Vortex the bottle for 30 sec.

2. Place on the benchtop at room temperature for 30 min.

3. Examine the reagent for precipitate (look into the top of the bottle).

4. If precipitate is still present, vortex again for 30 sec.

5. Pour Axiom Wash A into the appropriate reagent reservoir and leave on the benchtop until ready to use.

To prepare the Stabilize Diluent:

If crystals are observed in the Axiom Stabilize Diluent:

1. Vortex and Spin.

2. Look for precipitate. If any, warm tube to room temperature and vortex again.

Preparing Axiom Ligate BufferWhite precipitate is sometimes observed when the Axiom Ligate Buffer is thawed.

Note: The presence of some precipitate is okay and will not adversely impact assay performance. Follow the instructions below to attempt to resuspend a majority of precipitate before use.

To prepare the Axiom Ligate Buffer:

1. Place on the benchtop at room temperature for 30 min. This bottle can also be thawed in a dish with room temperature Millipore water.

2. Examine the buffer for precipitate (look into the top of the bottle).

3. If precipitate is present, vortex the bottle for 30 sec.

4. Re-examine the buffer for precipitate.

5. If precipitate is still present, warm the bottle with your hands and vortex again for 30 sec.

6. If precipitate is still present after hand warming proceed with the protocol below.

7. Pour the Axiom Ligate Buffer into the appropriate reagent reservoir and leave on the benchtop until ready to use.

Prepare the remaining reagents

To prepare the remaining reagents for GeneTitan MC Instrument Plate Preparation:

1. Leave the Axiom Ligate Enzyme at –20°C until ready to use.

2. Thaw the following reagents from Module 4-1 at –20°C on the benchtop at room temperature, then vortex, pulse-spin, and place on ice:

• Axiom Ligate Soln 1

• Axiom Probe Mix 1

• Axiom Stabilize Soln

• Axiom Stain Buffer

3. Prepare the remaining reagents from Module 4-2 as follows:

a. Gently flick each tube 2 to 3 times to mix, then pulse-spin.

b. Place reagents on ice, except for the Axiom Hold Buffer, Axiom Ligate Soln 2 and Axiom Water— leave these reagents on the benchtop at room temperature until ready to use.



3. Prepare the Sample Plate (if stored at –20°C) and the array plate

To prepare samples that were stored at –20°C:

1. Warm the Hyb Ready Plate at room temperature for 5 minutes. It is not necessary to equilibrate the plate for longer duration.

2. Make sure the Hyb Ready Plate is sealed well. If the plate is not sealed well:

a. Spin the plate and carefully remove the old seal.

b. If there is condensation on the top of the plate, blot dry gently with a Kimwipe.

c. Use a fresh seal and tightly reseal the plate.

3. Vortex the Hyb Ready Plate briefly, then pulse-spin to 1000 rpm.

4. Place the Hyb Ready Plate at room temperature.

To prepare the array plate:

1. Warm up the array plate on the benchtop before setting up hybridization on the GeneTitan MC Instrument.

a. Leave the array plate in the pouch at room temperature, for a minimum of 25 minutes, before opening and loading on the GeneTitan MC Instrument to allow the plate to come to room temperature.

b. At the end of the array warm up time, open the pouch and scan the array plate barcode into the Batch Registration file (see ̋ Stage 1: Create and upload Batch Registration fileʺ on page 144).

4. Prepare the GeneTitan™ MC Instrument

Before your samples are being denatured and/or the Biomek FXP Target Prep Express is preparing reagent trays, ensure that the GeneTitan MC Instrument is ready for use.

One or more of the following steps may need to be performed:

1. Launch AGCC and select AGCC GeneTitan Control.

2. Upload your sample registration file now, if you have not done so (see ʺStage 1: Create and upload Batch Registration fileʺ on page 144).

If you do not upload your samples before scanning the array plate barcode, the software will assign names to your samples.

3. On the AGCC GeneTitan Instrument Control, select the System Setup tab (Figure 53).

4. Configure the software as follows:

a. Setup Option: Hyb-Wash-Scan

Other options available are described under ʺSetup options for array plate processingʺ on page 140.

b. Click Next.

c. Plate Information:

• Barcode: Scan or manually enter the Axiom array plate barcode and click Next.

• Protocol Name: Select the protocol name and click Next.

5. Fill the Wash A, Wash B and Rinse bottles.

WARNING! Do not remove the array plate from the protective base or touch the surface of any arrays.



6. Empty the Waste bottle.

• After preparing the GeneTitan MC Instrument, perform steps 6a, 6b or 6c as appropriate for your workflow:

– Go to Step 6a on page 126: if you are denaturing the samples and transferring them to the hyb tray.

– Go to Step 6b on page 127: if you are preparing reagents for the Ligation-Wash-Stain.

– Go To Step 6c on page 127: if you are performing a 2-8 plate workflow, and you are denaturing samples for the 2nd plate and are preparing ligation reagents for the 1st plate. The samples for the 2nd Axiom array plate will be denatured and transferred into the Hyb tray. The Hyb tray will be placed in the GeneTitan MC Instrument with the array plate in preparation for hybridization. The Biomek FXP Target Prep Express will also prepare reagents for Ligation-Wash-Stain for the 1st array plate that was placed in the GeneTitan MC Instrument hybridization oven 24 hours ago.

Figure 53 Setup options for processing array plates.

System Setup tab

Select Hyb-Wash-Scan option



5. Run the Preparation for GeneTitan™ step

To run the Preparation for GeneTitan step:



3. Click the Run button.The Axiom 2.0 Target Prep run options window appears (Figure 54).

4. Select Preparation for GeneTitan™ and the sub-steps that you wish to run; then click OK.

Based upon your choices, the appropriate deck layout is displayed (Figure 55).

5. Deionize the GeneTitan stain trays. Refer to Appendix E, section entitled ʺDeionization procedure for GeneTitan™ trays and coversʺ on page 241 for the deionization procedure.






• Figure 58 on page 125—tube block

Note: Prior to placing the arched metal lid on the deck, be sure to clean the attached Microseal® P pad with 70% ethanol and dry it. Refer to the package insert for this product for further information on cleaning and replacement.

Figure 54 Software setup for Preparation for GeneTitan

You can run each step individually, or any combination of steps. If you do not select Prepare GeneTitan™ reagent trays, the deck layout will be different than the one shown in Figure 55.

• Select Denature Samples and Transfer denatured samples to hyb tray– if you are preparing to load the array plate into the GeneTitan

Instrument for Hybridization.• Select Prepare GeneTitan™ Reagent Trays

– if you are preparing to load the GeneTitan Instrument for Wash-Stain and Imaging.

• Select Denature Samples and Transfer denatured samples to hyb tray and Prepare GeneTitan™ Reagent trays

– if you are running an 8 plate workflow then you will need to select all three check boxes in some instances in preparation for the plate that will go into the GeneTitan Instrument for Hybridization.

• Select Prepare GeneTitan™ Reagent trays for the array plate that has completed hybridization and is ready to be processed in the GeneTitan Instrument for Ligation-Wash-Stain and Imaging.

IMPORTANT! It is important to deionize the GeneTitan MC Instrument trays to remove any static electricity on the trays. Static attraction by the trays may prevent the tray cover from being lifted up by the instrument.



Figure 55 Deck layout for Preparation for GeneTitan—denature, transfer to hyb tray, and GeneTitan plate preparation

The deck layout displayed is based upon the selections you make for this step.

IMPORTANT: Destatic the stain trays prior to placing them on the deck. See the section "Deionization procedure for GeneTitan™ trays and covers" on page 241.

Included in deck layout if the following options are selected:

• Denature samples• Transfer denatured samples to

Hyb Tray

Included in deck layout if Prepare GeneTitan™ reagent trays is selected.




Table 45 Labware and Reagent Locations on the Deck for GeneTitan Reagent Preparation

Position on deck


If Denature samples and Transfer denatured samples to Hyb Tray are selected, (no reagent tray preparation), only the labware listed below is required:

TL1 Biomek AP96 – P250 pipette tips (aqua) —

P1 Lid, arched metalClean before use (70% ethanol).

—

P2 Hyb Reaction Plate1 Hyb-ready samples

P3 Hyb Tray2 —

If Prepare GeneTitan™ reagent trays is selectd the labware listed below is required:

P4 Stain 12, 3 (second of two trays with Stain 1) —

P5 Stain 22, 3 —

P6 Lig2, 3 —

P7 Scan Tray on protective base* —

P8 Stbl2, 3 —

P9 Stain 12, 3 (first of two trays with Stain 1) —

P10 Tube block with one insert, room temperature See Figure 58 on page 125.

P11 Reservoirs in frame:• Quarter module, divided (1 and 2)• Quarter module, divided (3 and 4)• Quarter module, divided (5 and 6)

• Pour Axiom Water into reservoir 1• Pour Axiom Ligation Buffer into reservoir 5

1Axiom Water

3Empty

5Axiom LigateBuffer

2Empty

4Empty

6Empty




P12 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)

• Pour Axiom Hold Buffer into reservoir 1• Pour Axiom Wash A into reservoir 2• Leave reservoir 3 empty

Pelt_1 Reagent block, chilled to 4°C See Figure 57 on page 124

P13 Biomek Span P250 pipette tips (green) —

P14 Biomek Span P1000 pipette tips (yellow) —

1 For on-deck or off-deck denaturation using the TRobot 96, PTC-200, or PTC 0240, use the Bio-Rad Hard-Shell 96-well plate (Cat. No.HSP-9631). For off-deck denaturation using the Applied Biosystems 9700 or 2720 thermal cycler, use the Bio-Rad Hard-Shell Full-Height96-Well Semi-Skirted PCR Plate (Cat. No. HSS-9601) and place the semi-skirted PCR plate on top of a Costar Round Bottom plate (Cat.No. 3795) for “Transfer Denatured Samples to Hyb Tray”.

2 These trays are included in Axiom Array GeneTitan Consumables Kit (Cat. No. 901606).3 Label each of these stain trays as described above as described in "Labeling GeneTitan hybridization and reagent trays". For example,

label the Stain tray to be placed in P6 with the word “Lig”. This tray will contain the Ligation master mix.

Table 45 Labware and Reagent Locations on the Deck for GeneTitan Reagent Preparation (Continued)

Position on deck


1Axiom Hold

Buffer

2Axiom

Wash A

3Empty

Reservoir

IMPORTANT! It is critical that you write only on the proper location of the hyb tray (on the edge in front of wells A1 and B1) as illustrated in Figure 18 on page 55 or on the proper location of the stain/reagent trays (on the edge in front of wells A1 to C1) as illustrated in Figure 19 on page 56. Do NOT write on any other side, as this can interfere with sensors inside of the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.




Figure 57 Placement of reagents on chilled reagent block for GeneTitan Reagent Tray Preparation

IMPORTANT:


Axiom Ligate

Enzyme

Axiom Probe Mix 1

Axiom Probe Mix 2

Axiom Ligate Soln 1

Axiom Stain 2-B

Axiom Stain Buffer

Axiom Stabilize

Soln

Axiom Stabilize Diluent

Axiom Stain 2-A

Axiom Stain 1-B

Axiom Stain 1-A




A1

A1




Note: If the physical deck does not match the Deck Layout window exactly (Figure 55 on page 121), either modify the physical deck to match exactly or choose Abort in the Deck Layout window.

8. Click OK to continue the step.




Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.

If the Denature Samples option is selected, the Biomek FXP Target Prep Express places the samples onto the thermal cycler and runs the denaturation program. If Prepare GeneTitan™ reagent trays is also selected, reagent tray preparation for the GeneTitan MC Instrument starts while the samples are being denatured.

Figure 58 Placement of reagents on room temperature tube block

AxiomLigationSoln 2

A1



6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays

If you selected Denature samples and Transfer denatured samples to Hyb tray in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 120 and Figure 54 on page 120), use the following instructions:

1. Once the GeneTitan MC Instrument is ready, return to the Biomek FXP Target Prep Express click OK (prompt shown in Figure 59 above).

The denatured samples are then taken off the thermal cycler and are transferred to the hyb tray.

• Ensure that there are no air bubbles present in the hyb tray. Puncture any air bubbles that you see using a pipette tip.

2. Transfer the hyb tray to the GeneTitan MC Instrument and load. Refer to the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 134 for the proper way of loading the array plate and the hyb tray.

3. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 60).



• Clean the Microseal P Pad by wiping with 70% EtOH and dry.

Refer to the package insert for this product for further information on cleaning and replacement.

Figure 59 Prompt indicating denaturation is complete.

IMPORTANT! Immediately load the array plate and hyb tray into the GeneTitan MC Instrument.

Figure 60 Instructions displayed at the end of Step 4.



6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays

If you selected Prepare GeneTitan™ Reagent trays in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 120 and Figure 54 on page 120), use the following instructions:

1. Once the prompt shown in Figure 60 appears, remove the reagent trays and scan tray with Hold Buffer from the deck and cover with the appropriate lids.

2. Examine each tray to ensure that:

• All of the wells have been filled. If any wells do not contain reagents, then manually add reagents to these wells.

• There are no air bubbles present. Puncture any air bubbles that you see using a pipette tip.

3. Transfer the reagent trays, scan tray to the GeneTitan MC Instrument and load. Refer to the section ʺStage 3: Ligate, Wash, Stain, and Scanʺ on page 160 to continue the process on the GeneTitan MC instrument.

4. Return to the Biomek FXP Target Prep Express and clear the deck (see Figure 53 on page 119).





6c. Complete Stage 4: Preparation for GeneTitan™ - Multiple Plate Workflow

If you selected all three options in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 120 and Figure 54 on page 120) and are preparing hyb tray and reagent trays in a 2+ plate workflow, use the following instructions:

The Hyb tray is prepared for a new Axiom array plate that will be loaded into the GeneTitan MC Instrument. The reagent trays are prepared for the Axiom array plate that is in the hybridization oven in the GeneTitan MC Instrument and is ready to move to the next stage of the process - Ligation, Wash-Stain and Scan.

Note: When using an off-deck thermal cycler please refer to the instructions for the ʺOff-deck thermal cycler optionʺ on page 110.

1. Once the reagent trays are prepared and sample denaturation is complete, the prompt in Figure 59 on page 126 is displayed (do not click OK yet). Remove the reagent trays and scan tray with Hold Buffer from the deck and cover with the appropriate lids.

2. Examine each tray to ensure that:

• All of the wells have been filled. If any wells do not contain reagents, then manually add reagents to these wells.

• There are no air bubbles present. Puncture any air bubbles that you see using a pipette tip.

IMPORTANT! Immediately load the reagent trays onto the GeneTitan MC Instrument. Do not leave denatured samples or reagent trays at room temperature for any length of time.



3. Transfer the reagent trays, scan tray and array plate to the GeneTitan MC Instrument and load.

4. Return to the Biomek FXP Target Prep Express and click OK at the prompt shown in Figure 59 on page 126.

The denatured samples are then taken off the thermal cycler and are transferred to the hyb tray.

5. Transfer the hyb tray to the GeneTitan MC Instrument and load. Refer to the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 134 for the proper way of loading.

6. Transfer the reagent trays, scan tray to the GeneTitan MC Instrument and load. Refer to the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 134 to continue the process on the GeneTitan MC Instrument.

7. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 60 on page 126).





IMPORTANT! Immediately load the reagent trays and the hyb tray onto the GeneTitan MC Instrument. Then load the array plate and hyb tray. Do not leave denatured samples or reagent trays at room temperature for any length of time.



Summary of Preparation for GeneTitan MC™ Instrument

Preparation for the GeneTitan MC Instrument

Span-8MC

Preparation for the GeneTitan MC Instrument – Page 1

Move Hyb Lid onto Hyb Rxn Plate

Hyb Lid Hyb Rxn Plate

Fill Scan Tray with Hold Buffer

Hold Buffer Reservoir Hold Buffer Plate

150 μL/well

Continued on GeneTitan PrepPage 2


Prepare Stabilize Solution & Plate

Water Stabilize Soln

10727 μL 144.96 μL

(RT) (4oC)Stabilize Solution Reservoir

Stabilize Solution Plate

105 μL/well

Stabilize Diluent

(4oC)

1208 μL

Assay






Span-8MC



Transfer Wash A Buffer to Stain 1 & 2 Reservoirs

Stain 1 Reservoir Stain 2 Reservoir(RT)

Wash A Buffer

22233.6 μL 11596.8 μL

Continued from GeneTitan PrepPage 1


Transfer Stain Buffer to Stain 1 & 2 Reservoirs

Stain 1 Reservoir Stain 2 Reservoir(4oC)

Stain Buffer

463.2 μL 241.6 μL

Transfer Stains 1-A & 1-B to Stain 1 ReservoirStain 1-A Stain 1-B

231.6 μL 231.6 μL

(4oC) (4oC)Stain 1 Reservoir




Span-8MC




Transfer Stains 2-A & 2-B to Stain 2 Reservoir

Stain 2-A Stain 2-B

120.8 μL 120.8 μL

(4oC) (4oC)Stain 2 Reservoir


Prepare Stain 1 Plates

Stain 1 Plate 1 Stain 1 Plate 2

Stain 1 Reservoir

105 μL/well 105 μL/well





Span-8MC

Prepare Stain 2 PlateStain 2 Reservoir Stain 2 Plate

105 μL/well




Prepare Ligate Solution & Distribution Plate

Ligate Buffer Ligate Enzyme

7610.4 μL 181.2 μL

(RT) (4oC)MM Reservoir

Ligate Plate

105 μL/well

Ligate Soln 1

(4oC) 1510 μL

Ligate Soln 2

362.4 μL (RT)

1208 μL 1208 μL (4oC)(4oC)

Probe Mix 1 Probe Mix 2




Incubate Hybridization Plate in TRobot

Hyb Rxn Plate + LidTRobot

Incubate:

1) 10 min. @ 95oC 2) 3 min. @ 48oC




Span-8MC


Unload Hybridization Plate from TRobotHyb Rxn Plate + Lid

Position P2

Remove Lid from Hybridization Plate

Position P2 Position P1

Process Hybridization Trayon the GeneTitan Instrument

Transfer Samples to Hybridization TrayHyb Rxn Plate Hyb Tray

105 μL/well





5 Array processing with theGeneTitan™ MC Instrument

The Axiom™ 2.0 Assay is designed for processing 96 samples at a time on Axiom™ Genome-Wide and Custom myDesign™ array plates. The protocol is performed in two sets of steps:

• Target Preparation: Automated target prep, performed with the Biomek FXP Target Prep Express.

• Array Processing: performed on the GeneTitan Multi-Channel (MC) Instrument.

This chapter includes instructions for Part 2: Array Processing.

Before using the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Stage 1: Create and upload Batch Registration file. . . . . . . . . . . . . . . . . . . . . . . . 144

Stage 2: Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Status window prompts and actions required . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Stage 3: Ligate, Wash, Stain, and Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Continuing the workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Shutting down the GeneTitan™ MC Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . 170

Before using the GeneTitan™ MC Instrument

Note: For optimal GeneTitan MC Instrument performance, the maximum relative humidity must be 80% for temperatures up to 75.2°F (24°C) and a minimum humidity of 30 ±7% relative humidity. Operating outside the working environment specifications leads to higher static levels and results in the evaporation of reagents from stain trays.

Proper tray alignment and loading

Proper alignment and loading of plates, covers and trays is critical when using the GeneTitan MC Instrument. Each plate, cover and tray has one notched corner. The notched corner of plates, trays, covers and bases must be in vertical alignment with each other, and placed in position A1 per the Tray Alignment guide inside each GeneTitan MC Instrument drawer (Figure 61 and Figure 62 on page 136).

Note: Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading onto the GeneTitan MC Instrument.


CAUTION! Take care not to damage the consumables or bend the blue cover posts or scan tray posts.


Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument 5

Note: The instrument control software will display a warning if it detects a problem during the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and Rinse bottles should be replaced if the software displays such a warning. Refer to Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 247 for the message displayed to the user and the procedure for replacing the filters.

Figure 61 Proper alignment and loading of plates, covers and trays in the GeneTitan MC Instrument

Plates and trays must be seated in this rectangular recess.

The notched corner of all plates, bases, and covers and must be seated in this corner of the drawer per

Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading.

Notched corner of array plate aligned with notched corner of blue base.

IMPORTANT! Remove the plastic protective shipping tray cover. 1

2

3


1

2

344

5

5

6

7

7

6


Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument5

Figure 62 Array plate with protective blue base and the hyb tray aligned and properly loaded into drawer 6

Array Plate with Protective Blue Base

Hyb Tray

1

2

1 2

IMPORTANT! When you install the consumables, ensure that the fingers are retracted. Do not lay the consumables on top of the drawer fingers - this indicates that the instrument is not functioning correctly. Please notify your Field Service Engineer if the fingers do not retract automatically. You should place the trays into the instrument drawers when a drawer is fully extended by the instrument. The fingers are retracted when the drawer is open and are extended when the drawer is closed in order to restrain the consumable. See Figure 63 for an image showing the location of the tabs.

Figure 63 Photo identifying the location of drawer tabs

2

1

Drawer tab, or “finger” in back.

Drawer tab, or “finger” on right side.

1

2



Stain trays and covers


Proper labeling for hyb trays and reagent trays is described in:

• ʺLabeling for hyb traysʺ, below

• ʺLabeling for stain traysʺ on page 139

IMPORTANT! Always place the flat side of the cover against the stain tray.

Figure 64 Placement of covers on trays

Correct placement of cover on stain tray. Incorrect placement of cover on stain tray.

IMPORTANT! It is critical that you write only on the proper locations of the proper sides of hyb and stain trays. Do NOT write in any other location, as this can interfere with sensors inside the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.




Labeling for hyb trays

You may label the hyb tray on the front part of the short side of the tray, next to the notch at the left, as shown in Figure 65. The proper section for labeling is closest to the notched corner, corresponding to the A1 and B1 wells.

Figure 65 Labeling GeneTitan hyb trays

CAUTION! Writing on the wrong side of the Hyb tray, or on the wrong part of the long side, may interfere with the operation of sensors in the GeneTitan MC Instrument.


Notched corner of the hyb tray should face the front.

Label the hyb tray in this area.

1

2

3

1 2 3



Labeling for stain trays

You may label the stain trays on the left side of the front of the tray as shown in Figure 66. The correct side is closest to the notched corner, corresponding to the A1 through C1 wells.

Email and telephone notifications from the GeneTitan™ MC Instrument

We strongly recommend that you configure the Applied Biosystems™ GeneChip™ Command Console (AGCC) software to send you GeneTitan MC Instrument notifications. It is critical that you know when the instrument requires your attention—either for sample handling or troubleshooting. Rapid notification can lessen the risk of sample loss.

Notifications can be sent to email addresses and telephones. Refer to the Applied Biosystems™ GeneChip™ Command Console User Guide for instructions.

The types of notifications available will let you know when a process:

• Starts

• Completes

• Aborts

• Encounters an error

Figure 66 Labeling GeneTitan stain tray (stain tray shown with lid)


Notched corner of the stain tray should face the front.

Label the stain tray here.

1 2 3

1

2

3



GeneTitan™ MC Instrument lamp

The GeneTitan MC Instrument uses a xenon arc lamp system that is warranted to provide 500 hours of illumination for imaging the array at two wavelengths. The xenon lamp has a limited lifetime and needs to be replaced at regular intervals.

The GeneTitan Instrument Control software provides a timer that indicates the remaining useful life of the bulb and notifies you when it requires replacement. It is important to adhere to the warnings specified in the GeneTitan MC Instrument user guide.

Refer to the GeneTitan MC Instrument User Guide, Pub. No. 08-0308, or Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 249 of this user guide for details on replacing the lamp.

Refer to the GeneTitan MC Instrument User Guide, Pub. No. 08-0308, for the Lambda LS and Smart controller system. The Lamp and the controller should NEVER be switched ON or OFF manually. The GeneTitan MC Instrument control software manages the lamp activity and will switch the lamp ON and OFF as required. It takes 10 minutes to warm-up the lamp. In idle mode the lamp will remain ON for 2 hours before it is automatically switched OFF and if there are no more plates being transferred from the fluidics to the imaging station. This is by design and is intended behavior. Please do not try to save the lamp life by turning OFF the switch on the lamp.

Note: The power switch on the shutter box should be ON at all times. The OPEN/CLOSE switch on the shutter box should be at AUTO position at all times.

Setup options for array plate processing

The processes (setup options) available for processing array plates are shown in Figure 67. A brief description of each option is given below.

Figure 67 Setup Options for Processing Array Plates

System Setup tab

Setup options

1

2

1

2



Hyb-Wash-ScanThis setup option enables you to hybridize, wash-ligate-stain-stabilize, and scan an array plate on the GeneTitan MC Instrument.

• Hyb: the array plate is moved to the hybridization oven inside the instrument. Each denatured sample in the hyb tray is hybridized to an array on the array plate.

– Duration for 96 samples = 23.5 hr

• Wash: samples on arrays are ligated, washed, stained and stabilized.

– Duration for 96 samples = ~5 hr


• Scan: The array plate is moved to the imaging device in the GeneTitan MC Instrument and each array is scanned.

– Duration for 96 samples = ~7.5 hr

Hyb-WashIf this setup option is selected, array plate processing will stop after the array has gone through fluidics processing. Use this option if an array plate cannot be scanned on the same GeneTitan MC Instrument as the one used for hybridization and fluidics processing.

If the array plate cannot be scanned immediately after the Hyb-Wash process is complete:

1. Wrap the array plate (in the scan tray with black protective base) in aluminum foil to protect from light.

No lid is required. Do not invert the plate stack. If inverted, the Hold Buffer will spill out of the tray. To prevent liquid spillage, try to keep the plate level when handling the plates. Do not touch the bottom optical surface of the scan tray.

2. Store at 4°C.

3. Scan the array plate within 3 days or less.

When ready to scan the array plate:

1. Keeping the plate protected from light, bring the plate to room temperature for ~20 min.

2. Remove the aluminum foil and load onto the GeneTitan MC Instrument.

Wash-ScanUse this option if:

• You wish to bypass the Hybridization step and perform only the Wash/Stain and Scan steps.

Note: It usually takes 25-30 minutes to warm up Wash B if this option is selected.




Wash-Scan-ResumeUse this option if:

• It was necessary to hybridize the array plate in an oven separate from the GeneTitan MC Instrument.

• Fluidics processing has been interrupted (e.g., a power failure occurs at your facility).

ScanUse this option:

• To rescan an entire array plate or specific arrays on a plate that failed to scan for reasons such as bubbles or gridding failure.

• If you have hybridized and performed the fluidics processes on a different GeneTitan MC Instrument than the one you will currently use for the scan, or at a different time.

Unload Plates Use this option to unload plates and trays from the instrument when:

• Array plate processing is complete.

• Array plate processing has been aborted.



Aborting a process If necessary, you can abort the processing of one or more array plates. Instructions and an example are shown below in Figure 68.

If the instrument aborts a process, you can retrieve the array plate and related consumables as described in Figure 68. An instrument-initiated abort may occur:

• Due to improper placement of plates.

• If the uninterrupted power supply (UPS) detects a long power interruption, draining the UPS to 75% power.

Figure 68 Manually aborting an array plate

To abort array plate processing:

1. Click the Stop button.2. Select the array plate that you want to abort.3. Click Abort.4. Click Yes.5. Wait until the status of the array plate in the

WorkFlow window changes from AbortRequest… to Aborted.

6. Once aborted, retrieve the array plate and other related consumables by:• Using Setup Option: Unload Plates• Loading a new array plate.

Exception: If reagents are loading, abort the plate using the Cancel button displayed in the reagent load step.

Note: If the gripper is required to complete the Abort process, the plate will remain in the “AbortRequest” state until the gripper becomes available.

1

2

3

4

5


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 1: Create and upload Batch Registration file5

Stage 1: Create and upload Batch Registration file

You must create and upload a Batch Registration file in the Applied Biosystems™ GeneChip™ Command Console software before you begin ̋ Stage 2: Hybridizationʺ on page 145 (example shown in Figure 69). This file contains information critical for:

• Data file generation during scanning

• Tracking the experimental results for each sample loaded onto an array plate

1. If you have not already created a batch registration file, create one now. (See Appendix D, ʺRegistering samples in GeneChip™ Command Console™ʺ on page 238 for detailed instructions.)

2. In AGCC, select the array plate format (96 samples) and open a batch registration file template.

3. Scan the array plate barcode into the yellow barcode field.

4. Enter a unique name for each sample and any additional information.

5. Save the file.

6. Upload the file.

IMPORTANT! It is very important to create and upload a batch registration file with your sample information prior to starting ʺStage 2: Hybridizationʺ on page 145.

Figure 69 Example of a Batch Registration file

Your specific information is populated here.


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization 5

Stage 2: Hybridization

Reagents required Reagents required

An Axiom Genome-Wide Human or non-Human array plate or an Axiom myDesign™ Genotyping 96-Array Plate is required for this step. Prior to inserting this plate into the GeneTitan MC Instrument for hybridization, the array plate should be brought to room temperature as described below:

Warm up the array plate on the benchtop before setting up hybridization on the GeneTitan MC Instrument.

1. Remove the array plate box from the 4°C refrigerator where it is stored.

2. Open the box and remove the pouch containing the array plate and protective base.

3. Leave the array plate in the pouch, unopened but placed on the bench for a minimum of 25 minutes before opening and loading on the GeneTitan MC Instrument to allow the plate to come to room temperature.

4. At the end of the array warm up time, open the pouch and scan the array plate barcode into the Batch Registration file (see ʺStage 1: Create and upload Batch Registration fileʺ on page 144).

A hybridization tray containing denatured samples (from Step 2 on page 126 of Chapter 4) is also required for this step. The samples should be denatured and transferred to the hyb tray only after the GeneTitan MC Instrument is ready for loading the hyb tray in the ʺLoad Axiom™ array plate and hyb tray onto the GeneTitan™ MC Instrumentʺ section on page 150.

Table 46 Reagents required from the Axiom 2.0 Reagent Kit

Reagent Module

Axiom Wash Buffer A(both bottles; 1L) Module 3,

Room TemperaturePart No. 901472

Axiom Wash Buffer B

Axiom Water

WARNING! Do not remove the array plate from the protective base or touch the surface of any arrays.


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization5

Setup the instrument

To setup the instrument:

1. Launch AGCC Launcher and select AGCC GeneTitan Control (Figure 70).

The system initializes. After initialization, the System Status tab is selected and the status of the Hybridization Oven is displayed at the bottom of the Log window. The status should read: <Time of day> System Ready


IMPORTANT! Please do not close the scanner application by right-clicking on it and choosing the “Close” option. This will cause the scanner application to exit abnormally and cause undue delay in processing the next plate. The correct way to close the application is described in ʺShutting down the GeneTitan™ MC Instrumentʺ on page 170.

Figure 70 Launching AGCC and initializing the GeneTitan MC Instrument

System ready



2. Select the System Setup tab (Figure 71).

3. Configure the software as follows:

d. Setup Option: Hyb-Wash-Scan

Other options available are described under ʺSetup options for array plate processingʺ on page 140.

e. Click Next.

Figure 71 System Setup tab and the information displayed in this pane

Status: This field displays the actions that must be performed to prepare or unload the GeneTitan MC Instrument for the setup option that has been selected.

After each action you are instructed to click the Next button or to press the blinking blue Confirmation button located on the GeneTitan MC Instrument.

System Setup tab

Setup Option: The various options you can choose for processing Axiom array plates.

Workflow Steps: This field displays an overview of the user actions required to process an array plate based on the setup option selected.

Barcode: The array plate barcode. Can be scanned or entered manually.

Protocol Name: The protocol that GeneTitan MC Instrument will run. The list of protocols displayed is based on the first 6 digits of the array plate barcode. Only the protocols that are valid for the type of array plate loaded are displayed.

NOTE: If there is not enough disk space, a message is displayed.

• Delete or move .dat files to another location to free up enough disk space for the data that will be generated by eight Axiom array plates.

• One 96 Axiom array plate requires ~80 GB



f. Plate Information:

• Barcode: Scan or manually enter the Axiom array plate barcode and click Next.

The first six characters of the barcode identify the type of plate being loaded, the protocol GeneTitan MC Instrument will use to process the plate, and the imaging device parameters required for this type of plate.

• Protocol Name: Select the protocol name and click Next.

The system reads the first 6 digits of the array plate barcode to determine which protocols can be run for the type of array plate that has been loaded. Only valid protocols are displayed.

4. Complete the remaining workflow steps as follows:

a. Refill bottles with buffer (Figure 73 on page 149).Fill these bottles:

• Wash A: fill with Axiom Wash Buffer A—keep at 2L full

• Wash B: fill with Axiom Wash Buffer B—Use all 600 mL of Wash B from the reagent kit per Axiom plate. Fill to 1L mark when processing two plates on the same day.

• Rinse: fill with Axiom Water—keep at 1L full f

Figure 72 Barcode error message

IMPORTANT! • Always ensure that the GeneTitan bottles containing Wash A and Rinse are above

the 50% mark when setting up the system to process an Axiom array plate. All 600 mL of the Wash buffer B from the Axiom 2.0 reagent Kit should be emptied into the GeneTitan Wash B bottle when setting up the system to process a plate. This ensures that the GeneTitan Wash B bottle is filled to more than the requisite 35% of Wash B bottle volume. Also, do not overfill the bottles. Fill Wash Buffer B and Rinse bottles to the 1L mark only. Wash A keep at 2L. We strongly recommend refilling these bottles every time you are prompted to do so.

If the volume in any of these bottles becomes too low during a run, a message is displayed (see Chapter 8, ʺTroubleshootingʺ on page 215). However, even if you fill the bottle at this time, the instrument may not be able to successfully complete the step that was in progress.

• Wash B—if you intend to load two array plates on the same day, fill the Wash B bottle to the 1L mark (use both bottles from the Axiom 2.0 Reagent Kit).

If this error message is displayed:

• Ensure that the library files for the type of array plate you are using are correctly installed.

• Try manually entering the array plate barcode.• Library files must be installed prior to launching the GeneTitan MC Instrument. If a

library file must be installed, exit the GeneTitan MC Instrument, install libraries and relaunch the GeneTitan MC Instrument.



b. Empty the waste bottle.

c. Press the Confirmation button on GeneTitan MC Instrument to continue. A fluidics check is run (~1 min).

d. Empty trash bin

• Open the trash bin and empty.

If already empty, the trash bin remains locked and the Status pane reads “Trash bin is empty.”

• Press the Confirmation button to continue.

e. Remove consumable trays and plates

• Remove used trays and plates when drawers open.

• If no consumables to remove, the Status window reads “Drawers are empty.”


f. Continue to ̋ Load Axiom™ array plate and hyb tray onto the GeneTitan™ MC Instrumentʺ on page 150.

Figure 73 Example of the remaining workflow steps

Workflow step

Specific instructions for each workflow step



Load Axiom™ array plate and hyb tray onto the GeneTitan™ MC Instrument

The System Layout pane indicates the position of the various trays in each drawer during a GeneTitan MC Instrument run at maximum throughput. This pane does not change as plates are loaded or removed (Figure 74).

To load an Axiom array plate and hyb tray onto GeneTitan MC Instrument:

1. When drawer 6 opens, load the array plate and hyb tray as follows:

a. Examine the wells of the hyb tray for bubbles; puncture any bubbles with a pipette tip.

b. Load the hyb tray on the right side of the drawer (Figure 76 on page 151).

c. Remove the array plate and protective blue base from its package.

To avoid dust or other damage, leave the array plate packaged until ready to load onto the GeneTitan MC Instrument (Figure 75).

The array plate must be loaded on its protective blue base, as shown in Figure 76 on page 151. The clear plastic cover on top of the array plate SHOULD NOT be loaded in the GeneTitan MC Instrument. See Figure 61 on page 135 for more details on the correct way of loading the array plate.

Figure 74 System layout—location of plates inside the GeneTitan MC Instrument

IMPORTANT! Removing bubbles at this step greatly reduces the chance of bubbles under the arrays when the hyb tray and the Axiom array plate are clamped. Bubbles under an array can result in black spots on the array image.

Drawers showing contents.

Each line corresponds to a specific drawer number. In this example “Used Hyb Tray” is in the right side of Drawer 1, and “Hyb Tray” is the right side of Drawer 6.Note: Earlier versions of the software may show

as “Fix Tray” rather than “Stabilizing Tray”.

Right side of drawerLeft side of drawer



d. Load the array plate with the protective blue base on the left side of the drawer (Figure 76).

Figure 75 Array plate packaging

Figure 76 Array plate with protective blue base and the hyb tray properly loaded into drawer 6.

CAUTION! The notched corner of each plate, cover and tray must be aligned. When loading onto the GeneTitan MC Instrument, the notched edge plates, covers and trays must be aligned as indicated by the Tray Alignment guide in the drawer (Figure 76 on page 151).

The error message shown in may be displayed. Plate barcodes must face the internal barcode reader (back of the drawer). Improper tray positioning can cause the GeneTitan MC Instrument to crash, and can result in substantial damage to the instrument and loss of samples.

1

2

3


1

2

3

Array plate with protective blue base Hyb Tray



e. Press the Confirmation button.

When you load the array plate on the left side of the drawer, the internal bar code reader reads the barcode of the array plate and compares it with the barcode and the plate type specified in the Barcode and Plate Type fields on the Setup page. If the information is correct, the application allows you to proceed to the next step. If the instrument is unable to read the barcode, it will push the tray out and will prompt (Figure 77) you to load the correct plate with the proper orientation into the instrument (Figure 76).

– Click OK to retry and check the loading of the array plate; or

– Click Skip if the instrument has problems reading the barcode and after verifying that the trays have been placed in the proper orientation.

Figure 77 Barcode error message

IMPORTANT! Do not install a 3 plate stack of trays (Figure 78). Confirm that you have removed the clear plastic shipping cover as shown in Figure 61 on page 135.

Figure 78 Do Not install a three plate stack of trays



f. Select the arrays to scan (instructions in Figure 79).

By default, all arrays are selected.

2. Click Next, then click OK to begin processing the samples.

The array plate is placed on top of the hyb tray and clamped (now referred to as the plate stack).

The software starts the process for clamping the array plate to the hybridization tray. Press OK on the dialog shown in Figure 80 and wait for the drawer to open before retrieving the array plate and hybridization tray combo for inspection. The sandwich of the array plate and hybridization tray needs to be manually inspected before the array processing can begin. Once clamping is complete the dialog shown in Figure 81 on page 154 will be displayed. If you do not press OK in Figure 80 the dialog box will go away without intervention and Figure 81 on page 154 will be displayed.

Figure 79 Selecting which arrays to scan on an array plate

Figure 80 Clamping in progress notification

Default – all arrays are selected

Single array - click one box

Multiple arrays:

– Click one box– Hold down the Ctrl key– Click another box in the same column

Group of arrays:

– Click one box– Hold down the Shift key– Left-click and drag the mouse

Message in Status window.



3. When drawer 6 opens and the prompt in Figure 81 is displayed:

a. Remove the plate stack and gently press the two plates together at each clamping point.

Listen for a clicking sound which indicates that the plates are now clamped. No clicking sound indicates the plates are already clamped. (The figure below shows an example of an array plate and hybridization tray stack.)

Figure 81 Location of camping points on the array plate and hyb tray

Clamping Points on an Axiom Array Plate and Hyb Tray

Array Plate

Hyb Tray

Notched Corners

1 1

1 1

1 1

1

2

3

4

2

3

4



b. Inspect the bottom of the plate stack for bubbles under the arrays—do NOT invert the plates.

c. If bubbles are present, gently tap the plate until the bubbles move out from under the arrays—do NOT unclamp the plate stack.

d. Return the plate stack to the drawer, and press the Confirmation button to proceed.

The message below may be displayed again if plate orientation is incorrect or if the hyb tray barcode cannot be read. Click OK to proceed.



Load a second Axiom™ array plate and hyb tray onto the GeneTitan™ MC Instrument

When you can load a second array plate and hyb trayOnce processing begins, you have a specific period of time during which you can load another Axiom array plate and hyb tray. This period of time is displayed above the Hyb Oven Status pane (Figure 82). You cannot load another hyb tray before or after this period of time.

While the first plate is in the oven, you can load another plate if the time spacing requirement is met. This is to ensure that the second plate does not have to wait for system resources in its workflow. The time spacing is roughly equal to the longer of the wash-stain or scan time of the first plate.

IMPORTANT! You must load the next array plate and hyb tray during the period of time displayed above the Hyb Oven Status. You cannot load another hyb tray before or after this period of time. You will have to wait until the current process is finished which will result in disruption of the eight plate workflow and fewer than eight plates processed per week.

Figure 82 Loading a second hyb tray based on hybridization oven status information

This pane displays the period of time during which another array plate and hyb tray can be loaded.

Additional plates cannot be loaded before or after this period of time while the instrument is operating.

In this example the system is currently available.

Position of plate stack in the hybridization oven.

Position 1 - left side of oven

Position 2 - right side of oven

Oven Temperature.

• Green indicates the current oven temperature is within the target temperature range.

• Yellow indicates oven temperature outside of target temperature range.

1

2

3

1

2

3



1. Select the System Setup tab.

2. Load an Axiom array plate and hyb tray in the same manner that you loaded the previous plate and tray.

a. Scan or manually enter the Axiom array plate barcode, then click Next.

b. Load the Axiom array plate with the blue base (without the cover) and the hyb tray (as shown in Figure 76 on page 151), then press the Confirmation button.

c. Select the arrays to scan, then click Next.

d. Ensure that the plates are clamped securely when prompted, then press the Confirmation button.

e. Click OK when prompted to resume plate processing.

Select the System Status tab to view Axiom array plate status in the WorkFlow window (Figure 83).

Figure 83 Example of the workflow window when two plates are loaded and are in the hybridization oven

Location: Left and Right positions = the position of the scan tray in drawer 2 (left or right side of the drawer).

1

1


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStatus window prompts and actions required5

Status window prompts and actions required

As a part of normal GeneTitan MC Instrument operations you may see the following status prompts. Table 47 explains the necessary actions required.

Table 47 Refilling buffer bottles and emptying the waste bottle

Status window prompt Action required Receptacle – Reagent

Buffer bottles have been depressurized. Please refill buffer into the bottles. Empty the waste bottle.

• Replenish the fluid in Wash Bottles A and B, and the Rinse bottle.1

• Empty the Waste Bottle.• Press the Confirmation button to

continue.

• Wash Bottle A – fill with Axiom Wash Buffer A up to 2L.

• Wash Bottle B – fill with Axiom Wash Buffer B to the 1L mark.

• Rinse – fill with Axiom Water to the 1L mark.

Do not overfill these bottles.

1 Every time you are prompted to refill the buffer bottles, the system runs a fluidics check (duration ~1 min).

Table 48 Emptying the trash bin

Status window prompt Action required Receptacle – Reagent

Empty trash bin • Open and empty the trash bin.• Press the Confirmation button to

continue.

Note: If the trash bin is empty, you will not be able to open it. Continue the process by pressing the blue confirmation button

—


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStatus window prompts and actions required 5

Table 49 Loading the array plate and hyb tray; barcode error messages

Status window prompt Action required Reagent – Receptacle

Load array plate tray on [Left/Right] side of drawer. Load hyb tray without cover on [Left/Right] side of drawer.

Load the array plate with the blue base and the hyb tray in drawer 6.• IMPORTANT! The blue base must remain in “left side

HTA in” even when empty.• IMPORTANT! The trays must be positioned correctly. If

the trays are placed incorrectly, the software will display an error dialog box indicating the barcode could not be read.


• Hyb Tray loaded with denatured samples.

These messages are displayed if:• A plate has been loaded

improperly.• The bar code is missing

or obscured

Text version of the error message

WARNING! The system was not able to verify the array plate barcode.

Please verify that the tray on the left side of the drawer has a blue protective base and if applicable, an array plate, in the correct ORIENTATION. The right side of the drawer should contain a hyb tray, if applicable, in the correct ORIENTATION.

Details:

The consumable is either not the correct consumable, not loaded correctly, or its barcode is not readable. Proceeding with an incorrect or incorrectly loaded consumable can result in a loss of consumables, loss of samples and may require a Field Service Engineer to service the instrument.

Refer to the System Setup tab or the user guide provided with the Assay or AGCC for instructions on proper consumable placement.

Press the flashing blue confirmation button or...

Press OK, the GeneTitan MC Instrument will verify the barcode and orientation.

Press Skip, the GeneTitan MC Instrument will NOT verify the barcode and orientation. The barcode entered at registration will be used.

Table 50 Selecting which arrays to scan

Status window prompt Action required Reagent – Receptacle

Select arrays to scan • Accept the default (all arrays selected) if appropriate. Otherwise, select the arrays to be scanned.

• Click Next, then click OK to start processing.

—


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan5

Stage 3: Ligate, Wash, Stain, and Scan

Equipment, consumables, and reagents required

Scan tray with Axiom Hold Buffer• Cover the tray by orienting the notched corner of the cover over the notched edge

of the tray and leave on the benchtop (no need to protect from light).

CAUTION! Do not remove the scan tray from its protective black base. Leave the scan tray in the base until loaded onto the GeneTitan MC Instrument. When handling the scan tray, the bottom glass surface of the tray should not be touched.

Notched corner of the cover is aligned with the notched corner of the scan tray.

Always leave the scan tray in its protective black base.

1

1

2

2


Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan 5

Proper installation of the GeneTitan™ tray consumables

It is very important that you load the GeneTitan tray consumables in the proper orientation. The barcode faces into the instrument (refer to Figure 84 and Figure 85).


Figure 84 You must rotate and load the trays so that the barcode faces into the instrument.

Figure 85 The proper loading of the GeneTitan Tray consumables is shown(the image shows the Stain Tray and the Stain Tray cover as an example).

Notch(This faces out and left)

Barcode(This faces BACK TO THE REAR of the instrument)

Turn the tray and cover combo so that the barcodes face BACK AND INTO the instrument and the notch faces OUT AND TO THE LEFT.

FRONT(OF INSTRUMENTFACING YOU)

Notch faces out and left.“For Research Use Only” faces out.

Barcode faces in and back.

FOR RESEARCHUSE ONLY

FOR RESEARCHUSE ONLY



Load trays onto the GeneTitan™ MC Instrument

To load trays onto the GeneTitan MC Instrument:

When hybridization of an Axiom array plate has finished, a message alerts you to resume the workflow setup. Press OK and the software takes you directly back to the System Setup tab.

This prompt to continue into reagent load occurs when the hyb is complete. “Estimated Time Remaining” displayed under “Hybridization Oven Status” may display a time remaining of 0 to 30 minutes when the prompt occurs.

The GeneTitan MC Instrument will allow reagent load to take place after either:

• the estimated time counts down to zero, or

• the actual real world hyb time (as indicated by the computer clock) indicates the hyb is complete.

Note: The time estimate displayed on some systems may lag due to high CPU utilization. The GeneTitan MC Instrument allows the workflow to synchronize with the system clock to compensate for this situation during the final half hour of the hyb time estimate. When this prompt to resume reagent loading is displayed to the user there is no need to wait for the estimated time to count down to zero.

Follow the prompts displayed to continue with staining, ligation, stabilizing and scanning.

1. Follow the prompts in the Status window.

a. Wash Bottles A and B, and the Rinse Bottle—refill as necessary (the system will prime itself again); Waste bottle—empty if necessary.

• Wash bottle A—2L

• Wash Bottle B and Rinse Bottle—fill to 1L mark only.

b. Empty the trash bin.

c. Remove consumable trays and plates as instructed, except for the blue base.Leave the blue array plate base in drawer 6 even though the base is empty.

2. Load consumable trays and plates as follows:

a. Follow the prompts in the Status window (load sequence and prompts in Table 51).

b. Once loaded, examine each cover for droplets of liquid.

c. If any liquid is present, remove the tray, clean the cover and top of the tray with Kimwipes, and reload the tray.

CAUTION! • Orient trays as indicated by the guide inside the drawer. Improper

orientation may cause the run to fail.

• Remove the protective black base from the scan tray immediately prior to loading Figure 86 on page 164).

• Examine each cover for droplets of liquid after loading. Liquid on the cover can result in capillary phenomenon. As a result, the tray may stick to the cover and be lifted out of place inside the instrument.



Table 51 Sequence for loading the trays with reagents

Loading sequence by

drawer number

Left Right

Note: If the software is unable to verify the barcode on the scan tray and the scan tray cover, the software will display the following error message.

2 Scan Tray with cover—do not load the protective black base(left side of drawer as indicated in Status window)

Figure 86 on page 164

3 Stain Tray with Stain 1 Ligation Tray


4 Stain Tray with Stain 2 Stabilization Tray with Stabilization Reagent




5 Stain Tray with Stain 1 Empty

Table 51 Sequence for loading the trays with reagents (Continued)

Loading sequence by

drawer number

Left Right

Figure 86 Scan tray loaded in drawer 2

Do NOT load the protective black base packaged with the scan tray.



IMPORTANT! When you load the plates, or trays, insert them under the tabs, or fingers, that may protrude into the stage. Confirm that the tray is not resting on these fingers.

1

1

1 Tab or “finger” in GeneTitan drawer.

Figure 87 Stain 1 tray (left) and Ligation tray (right) loaded in drawer 3



3. At the prompt, click Yes to load another Axiom array plate and hyb tray.Right or left position is determined by the position of Axiom array plates already in the GeneTitan MC Instrument.

4. Follow the prompts and:

a. Setup Option: select Setup Another Run, then click Next.

b. Scan or manually enter the Axiom array plate barcode, then click Next.

c. Select a protocol, then click Next.

Figure 88 Stain 2 tray (left) and Stabilization tray (right) loaded in drawer 4.

Figure 89 Stain 1 Tray loaded in drawer 5



d. When drawer 6 opens:

• Remove the blue base from the previous Axiom array plate.

• Load a new Axiom array plate and new blue base on the left; load a new hyb tray on the right.

• Press the Confirmation button.

e. Click OK when prompted in the Confirm Resume Processing message.

f. When drawer 6 opens, confirm that the plate stack is securely clamped, then press the Confirmation button.

The following is a description of array plate movements in the GeneTitan MC Instrument as users execute a multi-plate workflow.

1. The plate stack which has finished hybridization is moved from the Hyb oven to drawer 1 (temporarily).

2. The new plate stack in drawer 6 is moved to the Hyb oven.

3. The plate stack currently in drawer 1 (see Step 1) is moved to the unclamping station where it is unclamped and moved into the fluidics section of the GeneTitan MC Instrument.

Note: At the end of a Hyb-Wash-Scan run, all plate and tray covers should be in the trash.

Figure 90 is an example of how the System Status Workflow window appears when three Axiom array plates are being processed.



Figure 90 Example of the System Status window—three Axiom array plates are being processed

Workflow indicates the number of plates being processed and where they are in the instrument. In this example, three Axiom array plates are being processed: two in the hyb oven and one in fluidics.Estimated Completion Time is for the current process.

Status area: Current status indicates that another (4th) plate cannot be added to the GeneTitan hybridization oven because both oven slots are currently in use.

Estimated Time Remaining for fluidics is adjusted as necessary. Adjustments can be due to process interruptions such as a drawer being opened.

Step currently executing in fluidics.

1

1

2

2

3

4

3

4


Chapter 5 Array processing with the GeneTitan™ MC InstrumentContinuing the workflow 5

Continuing the workflow

Once a plate has gone through the fluidics stage of the process, it is moved to the imaging device.

When the scanning process begins, the window shown in Figure 91 is displayed. This window must remain open while Axiom array plates are being scanned.

CAUTION! • The Scan Control window must remain open while Axiom array plates are

being scanned. Closing this window will halt the scanning process. You can minimize this window if necessary without creating any interference to the imaging.

• Do not manually, or through the AGCC transfer utility, move any data associated with the current plate that is being processed/scanned. Transferring data will dramatically slow scanning and may cause the computer to freeze.

Figure 91 Scan Control window


Chapter 5 Array processing with the GeneTitan™ MC InstrumentShutting down the GeneTitan™ MC Instrument5

Shutting down the GeneTitan™ MC Instrument

This procedure assumes that all of the Axiom array plates loaded onto the GeneTitan MC Instrument have been processed.

To Shutdown the GeneTitan MC Instrument:

1. On the System Setup page, open the Setup Options drop-down menu and select Unload Plates.

2. Unload all of the consumables as prompted.

3. Power off the GeneTitan MC Instrument by opening Tools Shutdown from the menu.

4. Exit the Applied Biosystems™ GeneChip™ Command Console software if it does not close automatically.

Note: If the instrument is processing an array plate, the software will not allow you to shut down the system.

WARNING! Do not attempt to shut down the GeneTitan MC Instrument while array plates are being processed.


6 Processing eight PharmacoScan™

array plates per week

The PharmacoScan™ Assay on the Biomek FXP eight plate workflow allows array processing of eight sample plates per five-day work week while performing target preparation on eight more sample plates. Samples prepared one week can be hybridized the following week to continue the workflow. This chapter includes tables that present the timing of the steps required to perform this workflow in a five-day work week, nine hours per day. The workflow was validated under the following assumptions:

• Three FTEs; one dedicated mPCR operator, 2 operators for Beckman Biomek FXP Windows® 7 protocols

• Two validated thermal cyclers available in lab

• No T-robot on the Biomek FXP Instrument

This chapter assumes user familiarity with all procedures for target preparation and array processing using the GeneTitan™ MC and Biomek FXP instruments as described in Chapter 4, ʺMultiplex PCR and Target preparation on the Biomek FXP with Windows® 7ʺ and Chapter 5, ʺArray processing with the GeneTitan™ MC Instrumentʺ.

Overview of the eight plate workflow of the PharmacoScan™ Assay on the Biomek FXP

For continual processing of plates, a summary of the activities by week is:

• Week 1

– Target preparation—perform target preparation on eight sample plates to create eight Hyb Ready plates.

• Week 2 (and beyond)

– Array processing—hybridize the eight plates prepared in Week 1.

– Target preparation—perform target preparation on eight more sample plates; array processing for these Hyb Ready plates can be carried out the following week.

A scheme illustrating the timing required for the second week of this workflow is given in Figure 1 and Figure 2, and steps performed manually for mPCR and on the Biomek FXP and GeneTitan MC Instrument are listed in Table 1 and Table 2. Here and throughout the chapter, plates undergoing the target preparation on the Biomek FXP Instrument are given letters A through H while plates undergoing array processing on the GeneTitan MC Instrument are given numbers 1 through 8. Hyb ready plates prepared on the Biomek FXP Instrument can be processed on arrays in any order on the GeneTitan MC Instrument. More detailed day-by-day instructions are given in Table 7 to Table 14 and Figure 3 to Figure 7.

IMPORTANT! Experienced users and careful timing are critical for the successful execution of this workflow.


172 ol for Biomek FXP (Windows® 7) User Guide

Chapter 6 Processing eight PharmacoScan™ array plates per weekOverview of the eight plate workflow of the PharmacoScan™ Assay on the Biomek FXP6

Figu

Pla

Day 5

PM AM PM

Us

PharmacoScan™ Assay 96-Array Format Automated Protoc

re 1 Week 2: mPCR activities for PharmacoScan Assay simultaneous eight plate workflow

te #

A

B

C

D

E

F

G

H

Day 1 Day 2 Day 3 Day 4

AM PM AM PM AM PM AM

Multiplex PCR (mPCR) setup

mPCR gel QC

mPCR incubationFreeze

er Activities Background Activities

t PharmacoScan™ array plates per weekthe PharmacoScan™ Assay on the Biomek FXP 6

173

workflow

bation

e GeneTitan™ MC Instrument

g in the GeneTitan™ MC Instrument

neTitan™ MC Instrument

Day 5

PM AM PM

Chapter 6 Processing eighOverview of the eight plate workflow of

PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP (Windows® 7) User Guide

Figure 2 Week 2: Activities for Biomek FXP Target Prep Express and GeneTitan MC simultaneous eight plate

Hybridization Setup (Denature & Transfer to Hyb Tray)

DNA Amplification Setup

Amplification incu

Hybridization in th

Fragmentation & Precipitation

mPCR spike-in

Resuspension, Hyb prep, & QC

Off-deck Centrifugation & Drying Pellets

GeneTitan™ Reagent Tray Prep & Loading

Fluidics processin

Imaging in the Ge

Freeze

Off-deck QC

Plate #

A

B

C

D

E

F

G

H

User Activities Background Activities

1

2

3

4

5

6

7

8




Table 1 Overview of the manual mPCR and automated target preparation of the PharmacoScan™ Assay on the Biomek FXP

Day Activities Plates

1 • mPCR three plates of genomic DNA• Amplify eight plates of genomic DNA

• A, E, H• A–H

2 • mPCR three plates of genomic DNA• Fragment and precipitate three plates amplified on Day 1• Freeze five plates of amplified DNA for fragmentation later in

the week

• B, C, D• A, E, H• B, C, D, F, G

3 • mPCR two plates of genomic DNA• Fragment and precipitate two more plates of amplified DNA• Centrifuge/dry, resuspend, and QC three plates precipitated

on Day 2

• F, G• B, C• A, E, H

4 • Fragment and precipitate three remaining plates of amplified DNA

• Centrifuge/dry and freeze pellets for two plates precipitated on Day 3

• D, F, G

• B, C

5 • Resuspend and QC two plates of frozen pellets from Day 4.• Centrifuge/dry, resuspend, and QC three plates precipitated

on Day 4

• B, C• D, F, G

IMPORTANT! Maintaining consistent timing during the setup of the GeneTitan™ MC Instrument is critical to containing the user interventions of the eight plate workflow within the work day. Once one process begins late, there is little opportunity to catch up until the end of the workflow.

Table 2 Overview of the Array Processing of the PharmacoScan™ Assay on the GeneTitan™ MC Instrument


1 • Hybridize two plates of denatured Hyb Ready samples • 1 and 2

2 • Load reagent trays in GTMC for fluidics and imaging of plates loaded on Day 1

• Hybridize two plates of denatured Hyb Ready samples

• 1 and 2

• 3 and 4



• 3 and 4

• 5 and 6



• 5 and 6

• 7 and 8


• 7 and 8


Chapter 6 Processing eight PharmacoScan™ array plates per weekOverview of the eight plate workflow of the PharmacoScan™ Assay on the Biomek FXP 6

Duration of assay steps

The duration of the manually performed Multiplex PCR activities is listed in Table 3.

The duration of steps involved in target preparation and array processing within the PharmacoScan Assay on the Biomek FXP is given in Table 4. This table also indicates where detailed instructions for each step can be found.

These step durations include:

• Deck setup, run time, and deck cleanup on the Biomek FXP

• Preparation and loading of the GeneTitan MC Instrument

• Off-deck steps of Centrifugation and Drying Pellets, DNA pellet resuspension on a microplate shaker, running the QC gel, and reading the OD plate

Times for thawing reagents are given separately in the detailed day-by-day instructions in the next section.

Table 3 Duration of PharmacoScan Multiplex PCR associated steps

Operation Time required

Multiplex PCR set up

Thaw mPCR reagents and prepare mPCR reactions (see Chapter 4 Stage 1A, step "2: Prepare the mPCR master mix")

60 min

mPCR incubation

Run mPCR program on thermal cycler (see Chapter 4 Stage 1A, step "4: Run the PharmacoScan mPCR thermal cycler protocol and proceed")

~3.5 hours1

1 Thermal cycler run time may vary slightly between models.

mPCR gel QC

Prepare samples for mPCR QC gel, and then Freeze reaction plate (see Chapter 4 Stage 1A, step "5: Store the mPCR reaction plate")

15 min

Load, run and image mPCR QC gel (see Appendix G, "mPCR quality control gel protocol")

30 min

mPCR spike-in

Thaw mPCR reaction plate (see Chapter 4 Stage 2 step "2. Thaw and prepare the amplified DNA samples and reagents")

20 min

Spike amplicon into corresponding WGA plate (see Chapter 4 Stage 2 step "3: mPCR Spike-in to Amplification Plate")

10 min



Table 4 Duration of individual operations involved in the PharmacoScan™ Assay for the Biomek FXP


Target Preparation steps

DNA Amplification (see Chapter 4, "Stage 1B: DNA amplification")• Followed by a 23 ±1 hr incubation in a 37°C oven

30 min

Fragmentation (see Chapter 4, "Stage 2: Fragmentation and purification")• Followed by overnight precipitation in a –20°C freezer

2 hours

Off-deck Centrifugation and Drying Pellets (see Chapter 4, Step "Stage 3: Centrifugation and drying pellets")

75 min

Resuspension, Hybridization Preparation, and QC (see Chapter 4, "Stage 4: Resuspension, Hybridization Preparation, and QC"• On-deck set up and processing and off-deck pellet resuspension on a

microplate shaker

45 min

Off-deck Sample QC: Fragmentation Gel and OD quantitation (see Appendix B and Appendix C)

45 min

Array processing steps

Prepare Hybridization Tray (see Chapter 4, Stage 5 step "6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays")• Sample Denaturation off-deck and Transfer to Hybridization Tray on

the Biomek FXP • Transfer reagent trays from Biomek to GeneTitan MC Instrument and

load Hybridization Tray into GeneTitan MC Instrument, begin hybridization

• 45 min

• 23.5—24 hrs

GeneTitan Reagent Tray Prep and Loading (see Chapter 4, Stage 5, step "6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays")• GeneTitan Reagent Preparation on the Biomek FXP • Transfer reagent trays from Biomek to GeneTitan MC Instrument and

load Reagent Trays into GeneTitan MC Instrument

60 min

Concurrent Hybridization Setup and Reagent Tray Prep and Loading (see Chapter 4, Stage 5 step "6c. Complete Stage 4: Preparation for GeneTitan™ - Multiple Plate Workflow")• GeneTitan Reagent Preparation on the Biomek FXP

• Load Reagent Trays into GeneTitan MC Instrument• Sample Denaturation and Transfer to Hybridization Tray on the

Biomek FXP

• Transfer reagent trays from Biomek to GeneTitan MC Instrument and load Hyb Tray into GeneTitan MC Instrument

60 min


Chapter 6 Processing eight PharmacoScan™ array plates per weekOverview of the eight plate workflow of the PharmacoScan™ Assay on the Biomek FXP 6

The hybridization time for the PharmacoScan Assay on the GeneTitan MC Instrument is 23.5 to 24 hrs (Table 5). This provides a 30-minute window during which the instrument control software prompts the user to load the reagents required for washing and staining. Loading the reagent trays into the GeneTitan MC Instrument at the mid-point of this 30-minute window is recommended to allow the wash procedures to begin 24 hrs after the start of hybridization. If at any time catch-up is required in the framework of the eight plate workflow, begin loading reagents at the beginning of this 30-minute window (i.e., immediately after prompting by the software).

Thawing frozen plates of amplified DNA

Five of the plates (B, C, D, F, and G) in the workflow are frozen at the conclusion of the 23 hr DNA Amplification period on Day 2. These plates should be thawed prior to performing the fragmentation step on Day 3 or 4 using the following instructions:

1. Place the deep-well plate in a small water bath. For example, pour Millipore water into a small tray. Place the frozen plate on the water in the tray.

2. Leave the plate in the water bath for ~1 hour until all wells have thawed, especially the middle wells.

3. Make sure to spin the plate down at 1000 rpm for 30 sec to get rid of the water from the water bath






Thawing plates with frozen pellets

Two of the plates (B and C) in the workflow are frozen at the conclusion of Centrifugation and Drying Pellets on Day 4. This plate must be pre-equilibrated at room temperature for at least 1.5 hours before proceeding with the resuspension and hybridization protocol using the following instructions:

1. Leave the plate on the bench for 1.5 hrs until all wells have equilibrated to room temperature.

2. If needed, pulse-spin the plate for 30 sec to bring down any droplets or condensation generated from thawing the plate.

Table 5 GeneTitan™ MC Instrument processing times in the PharmacoScan™ Assay for the Biomek FXP

Steps on the GeneTitan MC Instrument Time required

Hybridization in the GeneTitan MC Instrument oven at 48°C 23.5 to 24 hrs

Fluidics processing in the GeneTitan MC Instrument 5 hrs

Imaging 96-format array plates in the GeneTitan MC Instrument 7.5 hrs


Chapter 6 Processing eight PharmacoScan™ array plates per weekSimultaneous eight plate workflow: Target preparation and array processing of the PharmacoScan™ Assay on the Biomek FXP6

Simultaneous eight plate workflow: Target preparation and array processing of the PharmacoScan™ Assay on the Biomek FXP

Day 1 Multiplex PCR activities• Manually prepare 3 plates of mPCR (Plates A, E, and H) from genomic DNA.

• Run mPCR QC gel (Plates A, E, and H).

• Freeze mPCR reaction plates after PCR amplification (Plates A, E, and H).

Biomek FXP Target Prep Express activities• Sample Denaturation and Prepare Hybridization Tray on two plates of target

prepared the previous week (Plates 1 and 2).

• Amplify eight new plates of genomic DNA (Plates A through H).

GeneTitan™ MC Instrument activities• Load the hyb tray and array plate into the GeneTitan MC Instrument and begin

hybridization (Plates 1 and 2).

Reagent and plate handling• Begin thawing the amplification reagents, particularly the Axiom 2.0 Amp Soln

and Axiom Water, 60 min prior to the start of each reaction.

• Before each Hybridization Setup step, warm the array plate to room temperature unopened in its pouch for at least 25 min prior to loading the array plate and hyb tray into the GeneTitan MC Instrument.

Note: All amplifications should be set up on Day 1 to allow for a 23 ±1 hr amplification incubation for each plate.

Table 6 Day 1 activities for Multiplex PCR

Approximate times

Activity Plate Instrument Start time End time Duration

Thaw mPCR reagents and mPCR Reaction Set Up

A, E — 9:00 AM 10:00 AM 60 min

Run mPCR reactions A, E Thermal cycler1 10:00 AM 1:30 PM ~3.5 hrs


H — 12:30 PM 1:30 PM 60 min

Run mPCR reactions H Thermal cycler1 1:30 PM 5:00 PM ~3.5 hrs

Prepare samples for mPCR QC gel and Freeze mPCR reaction plate

A, E — 1:30 PM 2:00 PM 30 min

Load, run, and image mPCR QC gels A, E — 2:00 PM 2:30 PM 30 min2

Prepare samples for mPCR QC gel and Freeze mPCR reaction plate. Load, run, and image mPCR QC gel

H — 5:00 PM 5:45 PM 45 min

1 The thermal cycler must be an approved thermal cycler validated for the PharmacoScan Assay. See "Thermal cycler recommendationsand protocols" on page 25. Thermal cycler run time may vary slightly between models.

2 Assumes mPCR QC gels are run concurrently.


Chapter 6 Processing eight PharmacoScan™ array plates per weekSimultaneous eight plate workflow: Target preparation and array processing of the PharmacoScan™ Assay on the Biomek FXP 6

Table 7 Day 1 activities for Biomek FXP Target Prep Express and GeneTitan MC for simultaneous eight plate workflow

Approximate times

Activity1 Plate Instrument2 Start time End time Duration

Hybridization Setup• Denature• Transfer to Hyb Tray

1On- or off-line

Biomek

8:45 AM 9:30 AM 45 min

Hybridization 1 GTMC 9:30 AM 9:30 AM—Day 2 24 hrs

DNA Amplification A Biomek 10:00 AM 10:30 AM 30 min

DNA Amplification B Biomek 10:30 AM 11:00 AM 30 min

DNA Amplification C Biomek 11:00 AM 11:30 AM 30 min

DNA Amplification D Biomek 11:30 AM 12:00 PM 30 min

DNA Amplification E Biomek 12:00 PM 12:30 PM 30 min

DNA Amplification F Biomek 1:00 PM 1:30 PM 30 min

DNA Amplification G Biomek 1:30 PM 2:00 PM 30 min

DNA Amplification H Biomek 2:00 PM 2:30 PM 30 min


2On- or off-line

Biomek

4:15 PM 5:00 PM 45 min

Hybridization 2 GTMC 5:00 PM 5:00 PM—Day 2 24 hrs

1 See Table 4 on page 176 for sections of this user guide containing detailed descriptions for each activity.2 GTMC = GeneTitan Multi-Channel Instrument; Biomek = Beckman Biomek FXP Target Prep Express Instrument–Windows 7.




Figu

4 5 6

Pla

4 5 6

U

ation

incubation

in the GeneTitan™ MC Instrument

Gel

Hyb Setup


re 3 Day 1 activities for PharmacoScan Assay workflow

8 9 10 11 12 1 2 3

te #

A

E

H

F

G

H

2

A

B

C

D

E

1

8 9 10 11 12 1 2 3

Freeze

Prepare reagents required for the operation


mPCR gel QC

Warm array plate to Room Temperature



ser activities Background activities

mPCR incub

Amplification

Hybridization

mPCR

mPCR Gel

Gel

mPCR

Amp

Amp

Amp

Amp

Amp

Amp

Amp

Amp

Hyb Setup


Day 2 Multiplex PCR activities• Thaw 3 mPCR reaction plates from Day 1 for mPCR spike-in into corresponding

DNA amplification plate (Plates A, E, and H).

• Manually prepare 3 plates of mPCR (Plates B, C, and D) from genomic DNA.

• Run mPCR QC gel (Plates B, C, and D).

• Freeze mPCR reaction plates after PCR amplification (Plates B, C, and D).



• Prepare reagent trays for the GeneTitan MC Instrument (for Plates 1 and 2 already on the GeneTitan MC Instrument).

• Transfer the denatured samples and reagent trays to the GeneTitan MC Instrument.

• Fragment and precipitate Plates A, E and H.

• Freeze plates of amplified DNA (Plates B, C, D, F, and G) after each plate has incubated at 37°C for 23 hrs.

GeneTitan™ MC Instrument activities• Load reagent trays for Plates 1 and 2. These plates are moved from the hyb oven

to the fluidics area. After fluidics, the plates move to the imaging area of the instrument.

• Load the hyb tray and array plate into the GeneTitan MC Instrument and begin hybridization (Plates 3 and 4).

Reagent and plate handling• Begin thawing the fragmentation and precipitation reagents 30 min prior to the

start of each Fragmentation step. Thaw the corresponding mPCR reaction plate for amplicon spike-in at this time as well.

• Begin preparing the reagents 30 min prior to the start of each GeneTitan Reagent Tray Preparation and Loading step.

• Before each Hybridization Setup step, warm the array plate to room temperature unopened in its pouch for at least 25 min prior to loading the array plate and hyb tray into the GeneTitan MC Instrument.

Note:

• Plates A, E and H are fragmented and precipitated on Day 2 without freezing to preserve the 23 hr amplification incubation.

• Store Plates B, C, D, F, and G at –20°C following 23 hrs of amplification reaction incubation.

• Precipitation is carried out at –20°C overnight. If space allows, keep plates in a single layer during overnight precipitation.




Approximate times



B, C — 9:00 AM 10:00 AM 60 min

Run mPCR reactions B, C Thermal cycler1 10:00 AM 1:30 PM ~3.5 hours


D — 12:30 PM 1:30 PM 60 min

Run mPCR reactions D Thermal cycler1 1:30 PM 5:00 PM ~3.5 hours

Prepare samples for mPCR QC gel and Freeze mPCR reaction plates

B, C — 1:30 PM 2:00 PM 30 min

Load, run, and image mPCR QC gels

B, C — 2:00 PM 2:30 PM 30 min2

Prepare samples for mPCR QC gel and Freeze mPCR reaction plate. Load, run, and image mPCR QC gel

D — 5:00 PM 5:45 PM 45 min

Thaw mPCR Reaction Plate A — 9:00 AM 9:20 AM 20 min

mPCR spike-in into Amplification Plate

A — 9:20 AM 9:30 AM 10 min

Thaw mPCR Reaction Plate E — 11:00 AM 11:20 AM 20 min


E — 11:20 AM 11:30 AM 10 min

Thaw mPCR Reaction Plate H — 1:00 PM 1:20 PM 20 min


H — 1:20 PM 1:30 PM 10 min






Approximate times


Thaw reagents for GeneTitan Reagent Tray Prep

1 — 8:00 AM 8:30 AM 30 min

Concurrent Steps:• GeneTitan Reagent Tray

Prep and Loading• Denature & Hyb Setup

1

3

Biomek/GTMC

On- or off-line/Biomek

8:30 AM 9:30 AM 60 min



A Biomek 9:30 AM 11:30 AM 2 hours

Freeze (–20°C) at end of 23 hr DNA amplification

B Freezer 10:00 AM Overnight


C Freezer 10:30 AM Overnight


D Freezer 11:00 AM Overnight


E Biomek 11:30 AM 1:30 PM 2 hours


F Freezer 12:30 PM Overnight


G Freezer 1:00 PM Overnight


H Biomek 1:30 PM 3:30 PM 2 hours


2 — 3:30 PM 4:00 PM 30 min



2

4

Biomek/GTMC


4:00 PM 5:00 PM 60 min


1 See Table 4 for sections of the user guide containing detailed descriptions for each activity.2 GTMC = GeneTitan Multi-Channel Instrument; Biomek = Beckman Biomek FXP Target Prep Express Instrument–Windows 7.




Figu

5 6

P

5 6

Temperature

n™ MC Instrument


C Instrument

Gel

GT Reagent Prep

Hyb Setup



8 9 10 11 12 1 2 3 4

late #

B

C

D

F

G

H

2

A

B

C

D

E

1

8 9 10 11 12 1 2 3 4

3

4

FreezePrepare reagents required for the operation (including mPCR reaction plate)


mPCR gel QC

Warm array plate to Room


User activities

Background activities

mPCR incubation

Amplification incubation

Hybridization in the GeneTitaFragmentation & Precipitation

mPCR spike-in

Fluidics processing in the Ge

Imaging in the GeneTitan™ MGeneTitan™ Reagent Tray Prep

GeneTitan™ Instrument loading

mPCR

mPCR Gel

Gel

mPCR

Hyb Setup

Frag & Precip

Frag & Precip

Frag & Precip

GT Reagent Prep


Day 3 Multiplex PCR activities• Thaw 2 mPCR reaction plates from Day 2 for mPCR spike-in into its

corresponding DNA amplification plate (Plates B and C).

• Manually prepare 2 plates of mPCR (Plates F and G) from genomic DNA.

• Run mPCR QC gel (Plates F and G).

• Freeze mPCR reaction plates after PCR amplification (Plates F and G).




• Fragment and precipitate Plates B and C.

• Resuspend and QC Plates A, E, and H.

Off-deck activities• Centrifuge and dry Plates A, E, and H.

• Resuspension—Off-deck pellet resuspension on a microplate shaker.

• Sample QC—fragmentation gel and OD quantitation for Plates A, E, and H.






• Plates of amplified DNA that were frozen on Day 2 (Plates B and C) must be thawed before use in fragmentation; follow the procedure under ̋ Thawing frozen plates of amplified DNAʺ on page 177.

• Begin preparing the Resuspension reagents a minimum of 60 min prior to the start of the Resuspension step; Resuspension reagents for Plates A, E, and H can be prepared simultaneously to save time.


• Before each Hybridization Setup step, warm the array plate, which is unopened in its pouch, to room temperature for at least 25 min prior to loading the array plate and hyb tray into the GeneTitan MC Instrument.



Note:

• Following Centrifuging and Drying, Plates A, E, and H can be kept at room temperature prior to Resuspension and Hybridization Preparation on the Biomek FXP Instrument (seal the plates, then unseal prior to placing on the Biomek FXP deck). See Chapter 4, Stage 4, step ̋ 1. Preparing frozen pellets and Axiom Resusp Bufferʺ for further guidelines on storing pellets and warming plates of pellets if there will be a delay between pellet drying and resuspension.



Approximate times



F, G — 9:00 AM 10:00 AM 60 min

Run mPCR reactions F, G Thermal cycler1 10:00 AM 1:30 PM ~3.5 hours

Prepare samples for mPCR QC gel and Freeze mPCR reaction plates

F, G — 1:30 PM 2:00 PM 30 min

Load, run, and image mPCR QC gels

F, G — 2:00 PM 2:30 PM 30 min2

Thaw mPCR Reaction Plate B — 9:00 AM 9:20 AM 20 min


B — 9:20 AM 9:30 AM 10 min

Thaw mPCR Reaction Plate C — 11:00 AM 11:20 AM 20 min


C — 11:20 AM 11:30 AM 10 min




Activity1 Plate Instrument2 Approx. start time

Approx. end time Duration


3 — 8:00 AM 8:30 AM 30 min



3

5

Biomek/GTMC


8:30 AM 9:30 AM 60 min




Thaw DNA Amplification Plate B — 8:30 AM 9:30 AM 60 min

Fragmentation & Precipitation B Biomek 9:30 AM 11:30 AM 2 hours

Off-deck Centrifugation and Drying Pellets

A, E, H Centrifuge/Oven 9:45 AM 11:00 AM 75 min

Thaw DNA Amplification Plate C — 10:30 AM 11:30 AM 60 min

Fragmentation & Precipitation C Biomek 11:30 AM 1:30 PM 2 hours

Resuspension, Hyb prep, and QC

A Biomek 1:30 PM 2:15 PM 45 min

Off-deck QC A Spectrophotometer/E-Gel system

2:15 PM 3:00 PM 45 min


E Biomek 2:15 PM 3:00 PM 45 min

Off-deck QC E Spectrophotometer/E-Gel system

3:00 PM 3:45 PM 45 min


H Biomek 3:00 PM 3:45 PM 45 min


4 — 3:30 PM 4:00 PM 30 min

Off-deck QC H Spectrophotometer/E-Gel system

3:45 PM 4:30 PM 45 min



4

6

Biomek/GTMC


4:00 PM 5:00 PM 60 min




Activity1 Plate Instrument2 Approx. start time





Figu

4 5 6

Pla

4 5 6

te to Room Temperature

U




plification Plate

trument loading

GT Reagent Prep

Hyb Setup

eck QC

spension Off-Deck QC



8 9 10 11 12 1 2 3

te #

F

G

H

4

A

B

C

E

3

8 9 10 11 12 1 2 3

5

6

FreezePrepare reagents required for the operation (including mPCR reaction plate) Multiplex PCR (mPCR) setup

mPCR gel QC

Warm array pla


ser activities


mPCR incubation

Hybridization in th


mPCR spike-in

Fluidics processin

Imaging in the GeGeneTitan™ Reagent Tray Prep



Thaw DNA Am

GeneTitan™ Ins

Off-deck QC

mPCR

mPCR Gel

Gel

Hyb Setup

Frag & Precip

GT Reagent Prep

Centrifuge & Dry

Centrifuge & Dry

Centrifuge & Dry

Frag & Precip

Resuspension Off-Deck QC

Resuspension Off-D

Resu


Day 4 Multiplex PCR activities• Thaw 3 mPCR reaction plates from Day 2 and Day 3 for mPCR spike-in into its

corresponding DNA amplification plate (Plates D, F, and G).




• Fragment and precipitate Plates D, F and G.

Off-deck activities• Centrifuge and dry Plates B and C.

• Freeze Plate B and C after Centrifugation and Drying.

GeneTitan™ MC Instrument Activities • Load reagent trays for Plates 5 and 6. These plates are moved from the hyb oven



Reagent and plate handling• Begin thawing the Fragmentation and Precipitation reagents 30 min prior to the


• Plates of amplified DNA that were frozen on Day 2 (Plates D, F, and G) must be thawed before use in fragmentation; follow the procedure under ̋ Thawing frozen plates of amplified DNAʺ on page 177.


• Before each Hybridization Setup step, warm the array plate to room temperature which is unopened in its pouch for at least 25 min prior to loading the array plate and hyb tray into the GeneTitan MC Instrument.

Note:


• After being centrifuged and dried, Plate B and C are sealed and stored at –20°C.




Approximate times


Thaw mPCR Reaction Plate D — 9:00 AM 9:20 AM 20 min


D — 9:20 AM 9:30 AM 10 min

Thaw mPCR Reaction Plate F — 11:00 AM 11:20 AM 20 min


F — 11:20 AM 11:30 AM 10 min

Thaw mPCR Reaction Plate G — 1:00 PM 1:20 PM 20 min


G — 1:20 PM 1:30 PM 10 min


Approximate times



5 — 8:00 AM 8:30 AM 30 min

Thaw DNA Amplification Plate

D — 8:30 AM 9:30 AM 60 min



5

7

Biomek/GTMC


8:30 AM 9:30 AM 60 min



D Biomek 9:30 AM 11:30 AM 2 hours

Off-deck Centrifugation and Drying PelletsFreeze (–20°C) after Drying

B, C Centrifuge/Oven

Freezer

9:45 AM

11:00 AM

11:00 AM 75 min


F — 10:30 AM 11:30 AM 60 min


F Biomek 11:30 AM 1:30 PM 2 hours




G — 12:30 PM 1:30 PM 60 min


G Biomek 1:30 PM 3:30 PM 2 hours


6 — 3:30 PM 4:00 PM 30 min



6

8

Biomek/GTMC


4:00 PM 5:00 PM 60 min



Table 13 Day 4 activities for Biomek FXP Target Prep Express and GeneTitan MC for simultaneous eight plate workflow (Continued)

Approximate times





Figu

4 5 6

Pl

U

n Plate




loading

GT Reagent Prep

Hyb Setup



8 9 10 11 12 1 2 3

ate #

G

6

B

C

D

F

5

7

8

Freeze



ser activities


Prepare reagents required for the operation(including mPCR reaction plate)

mPCR spike-in

GeneTitan™ Reagent Tray Prep


Thaw DNA Amplificatio


Hybridization in th

Fluidics processin

Imaging in the Ge

GeneTitan™ Instrument

Hyb Setup

Frag & Precip

GT Reagent Prep

Centrifuge & Dry

Centrifuge & Dry

Frag & Precip

Frag & Precip


Day 5 Biomek FXP Target Prep Express activities• Prepare reagent trays for the GeneTitan MC Instrument (for Plates 7 and 8 already

on the GeneTitan MC Instrument).

• Resuspend and Sample QC Plates B, C, D, F, and G.

Off-deck activities• Centrifuge and dry Plates D, F, and G.


• Sample QC—fragmentation gel and OD quantitation for Plates B, C, D, F, and G.



Reagent and plate handling• Begin preparing the Resuspension reagents for a minimum of 60 min prior to the

start of the Resuspension step.

• The plate of pellets stored at –20°C (Plates B and C) must equilibrate to room temperature for 90 min prior to starting the Resuspension step.


Note: Following Centrifuging and Drying, Plates D, F, and G are sealed. Place plates F and G in a 4°C refrigerator until further processing later the same day. Plate D can be kept at room temperature prior to Resuspension on the Biomek FXP (seal the plates, then unseal prior to placing on the Biomek FXP deck). See Chapter 4, Stage 4, step ʺ1. Preparing frozen pellets and Axiom Resusp Bufferʺ for further guidelines on storing pellets and warming plates with DNA pellets if there will be a delay between pellet drying and Resuspension.




Approximate times


Warm Precipitation Plate to RT

B — 8:00 AM 9:30 AM 90 min


7 — 8:00 AM 8:30 AM 30 min

GeneTitan Reagent Tray Prep and Loading

7 Biomek/GTMC 8:30 AM 9:30 AM 60 min

Warm Precipitation Plate to RT

C — 8:45 AM 10:15 AM 90 min


B Biomek 9:30 AM 10:15 AM 45 min

Off-deck QC B Spectrophotometer/E-Gel system

10:15 AM 11:00 AM 45 min


D, F, G Plate centrifuge and oven

9:45 AM 11:00 AM 75 min


C Biomek 10:15 AM 11:00 AM 45 min

Off-deck QC C Spectrophotometer/E-Gel system

11:00 AM 11:45 AM 45 min


D Biomek 11:00 AM 11:45 AM 45 min

Off-deck QC D Spectrophotometer/E-Gel system

11:45 AM 12:30 PM 45 min


F Biomek 1:15 PM 2:00 PM 45 min

Off-deck QC F Spectrophotometer/E-Gel system

2:00 PM 2:45 PM 45 min


G Biomek 2:00 PM 2:45 PM 45 min

Off-deck QC G Spectrophotometer/E-Gel system

2:45 PM 3:30 PM 45 min


8 — 3:30 PM 4:00 PM 30 min


8 Biomek/GTMC 4:00 PM 5:00 PM 60 min



t PharmacoScan™ array plates per weekthe PharmacoScan™ Assay on the Biomek FXP 6

195

3 4 5 6


essing in the GeneTitan™ MC Instrument


GT Reagent Prep

ff-Deck QC

Chapter 6 Processing eighSimultaneous eight plate workflow: Target preparation and array processing of

PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP (Windows® 7) User Guide

Figure 7 Day 5 activities for PharmacoScan Assay workflow

8 9 10 11 12 1 2

Plate #

G

8

B

C

D

F

7


HybridizationWarm Precipitation Plate to room temperature

Fluidics proc

Imaging in th






Off-deck QC

GT Reagent Prep

Centrifuge & Dry

Centrifuge & Dry

Centrifuge & Dry





Resuspension O

7 Processing three PharmacoScan™

array plates per week

The PharmacoScan™ Assay on the Biomek FXP three plate workflow allows target preparation and array processing of three sample plates per week. This chapter includes tables that present the timing of the steps required to perform this workflow in a five-day work week, eight to nine hours per day. The workflow was validated under the following assumptions:

• One dedicated mPCR operator on Day 1, one operator for Beckman Biomek FXP Windows® 7 protocols on Day 1-Day 5

• Two validated thermal cyclers available in lab

• No T-robot on the Biomek FXP Instrument

This chapter assumes user familiarity with all procedures for target preparation and array processing using the GeneTitan™ MC and Biomek FXP instruments as described in Chapter 4, ʺMultiplex PCR and Target preparation on the Biomek FXP with Windows® 7ʺ and Chapter 5, ʺArray processing with the GeneTitan™ MC Instrumentʺ.

IMPORTANT! Experienced users and careful timing are critical for the successful execution of this workflow.

196 PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP User Guide

Chapter 7 Processing three PharmacoScan™ array plates per weekOverview of the three plate workflow of the PharmacoScan™ Assay on the Biomek FXP 7

Overview of the three plate workflow of the PharmacoScan™ Assay on the Biomek FXP

A scheme illustrating the timing required for the three plate workflow is given in Figure 1 and Figure 2. The three plates are referred to as Plates A, B and C for the target preparation and the GeneTitan Array Processing steps. The daily activities, which include manual mPCR, automated target preparation on the Biomek FXP, and array processing on the GeneTitan MC Instrument are listed in Table 1. Detailed day-by-day instructions are given in Table 6 to Table 12 and Figure 3 to Figure 7.

Figure 1 mPCR activities for PharmacoScan Assay three plate workflow

Plate #

A

B

C

Day 1

AM PM


mPCR gel QC

mPCR incubation

Freeze

User Activities

Background Activities

PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP User Guide 197

198 mated Protocol for Biomek FXP User Guide

Chapter 7 Processing three PharmacoScan™ array plates per weekOverview of the three plate workflow of the PharmacoScan™ Assay on the Biomek FXP7

Figu

ation




Pla

Day 5

PM AM PM

PharmacoScan™ Assay 96-Array Format Auto

re 2 Activities for Biomek FXP Target Prep Express and GeneTitan MC three plate workflow



Amplification incub

Hybridization in th


mPCR spike-in



GeneTitan™ Reagent Tray Prep & Loading

Fluidics processin

Imaging in the Ge

Freeze

Off-deck QC


te #

A

B

C





Table 1 Daily steps for PharmacoScan Assay three plate workflow


1 • Amplify three plates of genomic DNA• mPCR three plates of genomic DNA• mPCR Gel QC for three plates• Freeze mPCR reaction plates until needed for amplicon

spike-in step during Fragmentation

• A, B, C• A, B, C• A, B, C• A, B, C

2 • Fragment and precipitate two plates amplified on Day 1• Freeze one plate of amplified DNA for fragmentation on

Day 3

• A, B• C

3 • Centrifuge, dry, resuspend and QC two plates precipitated on Day 2

• Store one Hyb Ready plate in freezer• Thaw DNA Amplification Plate• Fragment and precipitate one plate of amplified DNA• Denature and begin hybridization for one plate on the

GeneTitan MC Instrument

• A, B

• B• C• C• A

4 • Centrifuge, dry, resuspend and QC plate precipitated on Day 3

• Denature and begin Hybridization for two plates on the GeneTitan MC Instrument

• GeneTitan Reagent Trays Preparation and Loading

• C

• B, C

• A

5 • GeneTitan Reagent Trays Preparation and Loading • B, C

IMPORTANT! Maintaining consistent timing during the setup of the GeneTitan™ MC Instrument is critical to containing the user interventions of the three plate workflow within the work day. Once one process begins late, there is little opportunity to catch up until the end of the workflow.


Chapter 7 Processing three PharmacoScan™ array plates per weekOverview of the three plate workflow of the PharmacoScan™ Assay on the Biomek FXP7

Duration of assay steps

The duration of the manually performed Multiplex PCR activities is listed in Table 2.

The duration of steps involved in target preparation and array processing within the PharmacoScan Assay on the Biomek FXP is given in Table 3. This table also indicates where detailed instructions for each step can be found.

These step durations include:

• Deck setup, run time, and deck cleanup on the Biomek FXP

• Preparation and loading of the GeneTitan MC Instrument

• Off-deck steps of Centrifugation and Drying Pellets, DNA pellet resuspension on a microplate shaker, running the QC gel, and reading the OD plate

Times for thawing reagents are given separately in the detailed day-by-day instructions in the next section.

Table 2 Duration of PharmacoScan Multiplex PCR associated steps


Multiplex PCR set up

Thaw mPCR reagents and prepare mPCR reactions (see Chapter 4 Stage 1A, step "2: Prepare the mPCR master mix")

60 min

mPCR incubation

Run mPCR program on thermal cycler (see Chapter 4 Stage 1A, step "4: Run the PharmacoScan mPCR thermal cycler protocol and proceed")

~3.5 hours*

* Thermal cycler run time may vary slightly between models.

mPCR gel QC

Prepare samples for mPCR QC gel, and then Freeze reaction plate (see Chapter 4 Stage 1A, step "5: Store the mPCR reaction plate")

15 min

Load, run and image mPCR QC gel (see Appendix G, "mPCR quality control gel protocol")

30 min

mPCR spike-in

Thaw mPCR reaction plate (see Chapter 4 Stage 2 step "2. Thaw and prepare the amplified DNA samples and reagents")

20 min

Spike amplicon into corresponding WGA plate (see Chapter 4 Stage 2 step "3: mPCR Spike-in to Amplification Plate")

10 min



Table 3 Duration of individual operations involved in the PharmacoScan™ Assay for the Biomek FXP


Target Preparation steps

DNA Amplification (see Chapter 4, "Stage 1B: DNA amplification")• Followed by a 23 ±1 hr incubation in a 37°C oven

30 min

Fragmentation (see Chapter 4, "Stage 2: Fragmentation and purification")• Followed by overnight precipitation in a –20°C freezer

2 hours

Off-deck Centrifugation and Drying Pellets (see Chapter 4, Step "Stage 3: Centrifugation and drying pellets")

75 min

Resuspension, Hybridization Preparation, and QC (see Chapter 4, "Stage 4: Resuspension, Hybridization Preparation, and QC"• On-deck set up and processing and off-deck pellet resuspension on a

microplate shaker

45 min

Off-deck Sample QC: Fragmentation Gel and OD quantitation (see Appendix B and Appendix C)

45 min

Array processing steps

Prepare Hybridization Tray (see Chapter 4, Stage 5 step "6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays")• Sample Denaturation off-deck and Transfer to Hybridization Tray on

the Biomek FXP • Transfer reagent trays from Biomek to GeneTitan MC Instrument and

load Hybridization Tray into GeneTitan MC Instrument, begin hybridization

• 45 min

• 23.5—24 hrs

GeneTitan Reagent Tray Prep and Loading (see Chapter 4, Stage 5, step "6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays")• GeneTitan Reagent Preparation on the Biomek FXP • Transfer reagent trays from Biomek to GeneTitan MC Instrument and

load Reagent Trays into GeneTitan MC Instrument

60 min


Chapter 7 Processing three PharmacoScan™ array plates per weekThree plate workflow: Target preparation and array processing of the PharmacoScan™ Assay on the Biomek FXP7

The hybridization time for the PharmacoScan Assay on the GeneTitan MC Instrument is 23.5 to 24 hrs (Table 4). This provides a 30-minute window during which the instrument control software prompts the user to load the reagents required for washing and staining. Loading the reagent trays into the GeneTitan MC Instrument at the mid-point of this 30-minute window is recommended to allow the wash procedures to begin 24 hrs after the start of hybridization. If at any time catch-up is required in the framework of the three plate workflow, begin loading reagents at the beginning of this 30-minute window (i.e., immediately after prompting by the software).

Thawing frozen plates of amplified DNA

One plate (C) in the workflow is frozen at the conclusion of the 23 hr DNA Amplification period on Day 2. This plate should be thawed prior to performing the fragmentation step on Day 3 using the following instructions:

1. Place the deep-well plate in a small water bath. For example, pour Millipore water into a small tray. Place the frozen plate on the water in the tray.

2. Leave the plate in the water bath for ~1 hour until all wells have thawed, especially the middle wells.

3. Make sure to spin the plate down at 1000 rpm for 30 sec to get rid of the water from the water bath






Three plate workflow: Target preparation and array processing of the PharmacoScan™ Assay on the Biomek FXP

Note: The Day 1 schedule is written for two people working together. One person sets up the whole genome amplification for all three plates on the Biomek FXP, and the other person sets up and runs the mPCR reactions for all three plates. When the mPCR run is complete, the mPCR QC gel can be run (optional), and then the plates are frozen until needed.

Genomic DNA sample for amplification should be prepared as described in Chapter 2, ʺGenomic DNA preparation and requirementsʺ on page 14.

Table 4 GeneTitan™ MC Instrument processing times in the PharmacoScan™ Assay for the Biomek FXP

Steps on the GeneTitan MC Instrument Time required

Hybridization in the GeneTitan MC Instrument oven at 48°C 23.5 to 24 hrs

Fluidics processing in the GeneTitan MC Instrument 5 hrs

Imaging 96-format array plates in the GeneTitan MC Instrument 7.5 hrs


Chapter 7 Processing three PharmacoScan™ array plates per weekThree plate workflow: Target preparation and array processing of the PharmacoScan™ Assay on the Biomek FXP 7

Day 1 Multiplex PCR activities• Manually prepare 3 plates of mPCR (Plates A, B, and C) from genomic DNA. Two

thermal cyclers are required.

• Run mPCR QC gel (Plates A, B, and C).

• Freeze mPCR reaction plates after PCR amplification (Plates A, B, and C).

Biomek FXP Target Prep Express activities• Amplify three plates of genomic DNA (Plates A, B, and C).

Reagent and plate handling• Begin thawing the amplification reagents, particularly the Axiom 2.0 Amp Soln

and Axiom Water, 60 min prior to the start of each reaction.

Note: All amplifications should be set up on Day 1 to allow for a 23 ±1 hr amplification incubation for each plate.


Approximate times



A, B — 9:00 AM 10:00 AM 60 min

Run mPCR reactions A, B Thermal cycler* 10:00 AM 1:30 PM ~3.5 hrs


C — 12:30 PM 1:30 PM 60 min

Run mPCR reactions C Thermal cycler1 1:30 PM 5:00 PM ~3.5 hrs

Prepare samples for mPCR QC gel and freeze mPCR reaction plate plate

A — 1:30 PM 1:45 PM 15 min

Prepare samples for mPCR QC gel and freeze mPCR reaction plate

B — 1:45 PM 2:00 PM 15 min

Load, run, and image mPCR QC gels A, B — 2:00 PM 2:30 PM 30 min†

Prepare samples for mPCR QC gel and freeze mPCR reaction plate

C — 5:00 PM 5:15 PM 15 min

Load, run, and image mPCR QC gel C — 5:15 PM 5:45 PM 30 min

* The thermal cycler must be an approved thermal cycler validated for the PharmacoScan Assay. See "Thermal cycler recommendationsand protocols" on page 25. Thermal cycler run time may vary slightly between models.

† Assumes mPCR QC gels are run concurrently.




es

ment–Windows 7.

nd time Duration

:00 AM 30 min

:00 PM 30 min

:00 PM 30 min

Figu

4 5 6

P

4 5 6

U

n

ubation

the GeneTitan™ MC Instrument

GelPrep


Table 6 Day 1 activities for Biomek FXP Target Prep Express for three plate workflow

Approximate tim

Activity*

* See Table 3 on page 201 for sections of this user guide containing detailed descriptions for each activity.

Plate Instrument†

† GTMC = GeneTitan Multi-Channel Instrument; Biomek = Beckman Biomek FXP Target Prep Express Instru

Start time E

DNA Amplification A Biomek 10:30 AM 11

DNA Amplification C Biomek 11:30 AM 12

DNA Amplification B Biomek 2:30 PM 3


8 9 10 11 12 1 2 3

late #

A

B

C

A

B

C

8 9 10 11 12 1 2 3

Freeze

Thaw mPCR reagents


mPCR gel QC

Prepare Reagents for DNA Amplification

Prepare samples for mPCR QC gel



mPCR incubatio

Amplification inc

Hybridization in

mPCR

mPCR Gel

Gel

mPCR

Amp

Amp

Amp

Prep

Prep


Day 2 Multiplex PCR activities• Thaw two mPCR reaction plates from Day 1 for mPCR spike-in into

corresponding DNA amplification plate (Plates A and B).

Biomek FXP Target Prep Express activities• Fragment and precipitate Plates A and B.

• Freeze plate of amplified DNA (Plate C) after plate has incubated at 37°C for 23 hrs.



Note:

• Plates A and B are fragmented and precipitated on Day 2 without freezing to preserve the 23 hr amplification incubation.

• Store Plate C at –20°C following 23 hrs of amplification reaction incubation.



Approximate times


Thaw mPCR Reaction Plate A — 9:30 AM 9:50 AM 20 min


A — 9:50 AM 10:00 AM 10 min

Thaw mPCR Reaction Plate B — 1:30 PM 1:50 PM 20 min


B — 1:50 PM 2:00 PM 10 min

Table 8 Day 2 activities for Biomek FXP Target Prep Express for three plate workflow

Approximate times

Activity* Plate Instrument† Start time End time Duration


A Biomek 10:00 AM 12:00 PM 2 hours


C Freezer 11:00 AM Overnight


B Biomek 2:00 PM 4:00 PM 2 hours

* See Table 3 for sections of the user guide containing detailed descriptions for each activity.† GTMC = GeneTitan Multi-Channel Instrument; Biomek = Beckman Biomek FXP Target Prep Express Instrument–Windows 7.




Figu

4 5 6

Pla

U

ation

Gel

ip



8 9 10 11 12 1 2 3

te #

y y y

A

B

C

Freeze


Amplification incub


Prepare reagents for Fragmentation (including mPCR reaction plate)

mPCR spike-in

Frag & Precip

Frag & Prec


Day 3 Multiplex PCR activities• Thaw mPCR reaction plates from Day 1 for mPCR spike-in into its corresponding

DNA amplification plate (Plate C).

Biomek FXP Target Prep Express activities• Resuspend and QC Plates A and B.

• Fragment and precipitate Plate C.

• Sample Denaturation and Prepare Hybridization Tray on Plate A.

• Store Hyb Ready Plate at –20°C until Hybridization on Day 4 (Plate B).

Off-deck activities• Centrifuge and dry Plates A and B.


• Sample QC—fragmentation gel and OD quantitation for Plates A and B.

GeneTitan™ MC Instrument activities• Load the hyb tray and array plate into the GeneTitan MC Instrument and begin

hybridization (Plate A).



• Plates of amplified DNA that were frozen on Day 2 (Plate C) must be thawed before use in fragmentation; follow the procedure under ʺThawing frozen plates of amplified DNAʺ on page 202.

• Begin preparing the Resuspension reagents a minimum of 60 min prior to the start of the Resuspension step.

• Before the Hybridization Setup step, warm the array plate, which is unopened in its pouch, to room temperature for at least 25 min prior to loading the array plate and hyb tray into the GeneTitan MC Instrument.

Note:

• Following Centrifuging and Drying, Plates A and B can be kept at room temperature prior to Resuspension and Hybridization Preparation on the Biomek FXP Instrument (seal the plates, then unseal prior to placing on the Biomek FXP deck). See Chapter 4, Stage 4, step ʺ1. Preparing frozen pellets and Axiom Resusp Bufferʺ for further guidelines on storing pellets and warming plates of pellets if there will be a delay between pellet drying and resuspension.





Approximate times


Thaw mPCR Reaction Plate C — 12:30 PM 12:50 PM 20 min


C — 12:50 PM 1:00 PM 10 min

Table 10 Day 3 activities for Biomek FXP Target Prep Express and GeneTitan MC for three plate workflow

Activity* Plate Instrument† Approx. start time



A, B Centrifuge/Oven 8:45 AM 10:00 AM 75 min

Resuspension and Hyb Plate prep

A Off-line, Biomek 10:00 AM 10:45 AM 45 min

Resuspension and Hyb Plate prep

B Off-line, Biomek 10:45 PM 11:30 PM 45 min

Off-deck QC A, B Spectrophotometer/E-Gel

11:30 AM 12:15 PM 45 min

Thaw DNA Amp Plate C — 12:00 PM 1:00 PM 60 min


C Biomek 1:00 PM 3:00 PM 2 hours


A On- or off-line/Biomek

4:15 PM 5:00 PM 45 min

Hybridization A GTMC 5:00 PM 5:00 PM—Day 4 24 hrs



e PharmacoScan™ array plates per weekthe PharmacoScan™ Assay on the Biomek FXP 7

209

3 4 5 6


Hyb Setup

Chapter 7 Processing threThree plate workflow: Target preparation and array processing of

PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP User Guide

Figure 5 Day 3 activities for PharmacoScan Assay workflow

8 9 10 11 12 1 2

Plate #

A

B

C



User activities Background activities

Hybridization


Prepare reagents required for the operation(including mPCR reaction plate)

mPCR spike-in




Off-deck QC

Centrifuge & Dry

Centrifuge & Dry

Frag & Precip




Day 4 Biomek FXP Target Prep Express activities• Sample Denaturation and Prepare Hybridization Tray (Plate B and C).

• Prepare reagent trays for the GeneTitan MC Instrument (for Plate A already on the GeneTitan MC Instrument).

• Resuspend and QC Plate C.

Off-deck activities• Centrifuge and dry Plate C.

• Resuspension—Off-deck pellet resuspension on a microplate shaker (Plate C).

• Sample QC—fragmentation gel and OD quantitation (Plate C).

GeneTitan™ MC Instrument Activities • Load reagent trays for Plate A. This plate is moved from the hyb oven to the

fluidics area. After fluidics, the plates move to the imaging area of the instrument.

• Load the hyb tray and array plate into the GeneTitan MC Instrument and begin hybridization (Plate C).

Reagent and plate handling• Begin preparing the resuspension reagents a minimum of 60 min prior to the start

of the Resuspension step.


• Before each Hybridization Setup step, warm the array plate to room temperature which is unopened in its pouch for at least 25 min prior to loading the array plate and hyb tray into the GeneTitan MC Instrument.

IMPORTANT! The GeneTitan reagent trays for array processing cannot be loaded until the array plate has finished hybridization, and they should not be prepared more than 1.5 hours before hybridization will finish. The GeneTitan reagent trays cannot be prepared ahead of time and stored.




Approximate times


Hybridization Setup:• Denature• Transfer to Hyb Tray

B On- or off-line/Biomek

8:45 AM 9:30 AM 45 min

Hybridization B GTMC 9:30 AM 9:30 AM—Day 5 24 hrs


C Centrifuge/Oven 9:30 AM 10:45 AM 75 min

Resuspension and Hyb Prep

C Biomek 10:45 AM 11:30 AM 45 min

Off-deck QC C Spectrophotometer/E-gel platform

11:30 AM 12:15 PM 45 min


A — 3:30 PM 4:00 PM 30 min



A

C

Biomek/GTMC


4:00 PM 5:00 PM 60 min

Hybridization C GTMC 5:00 PM 5:00 PM—Day 5 24 hrs





Figu

4 5 6

P

he GeneTitan™ MC Instrument


GT Reagent Prep

Hyb Setup



8 9 10 11 12 1 2 3

late #

A

B

C



User activities





Hybridization in t

Fluidics processin



Off-deck QC

Hyb Setup

Centrifuge & Dry Resuspension Off-Deck QC


Day 5 Biomek FXP Target Prep Express activities• Prepare reagent trays for the GeneTitan MC Instrument (for Plates B and C

already on the GeneTitan MC Instrument).

GeneTitan™ MC Instrument activities• Load reagent trays for Plates B and C. These plates are moved from the hyb oven


Reagent and plate handling• Begin preparing the reagents 30 min prior to the start of each GeneTitan Reagent

Tray Preparation and Loading step.


Approximate times



B — 8:00 AM 8:30 AM 30 min


B Biomek/GTMC 8:30 AM 9:30 AM 60 min


C — 3:30 PM 4:00 PM 30 min


C Biomek/GTMC 4:00 PM 5:00 PM 60 min





Figu

4 5 6

P

U

he GeneTitan™ MC Instrument



GT Reagent Prep



8 9 10 11 12 1 2 3

late #

B

C

ser Activities Background Activities

Hybridization in t

Fluidics processin

Imaging in the Ge

Thaw reagents for GeneTitan™ Reagent Tray Prep



GT Reagent Prep

8 Troubleshooting

Biomek FXP Target Prep Express

If a hardware problem is encountered while running the Axiom target preparation methods on the Biomek FXP Target Prep Express, you can do the following:

• Refer to these documents:

– Biomek® Liquid Handler User’s Manual, Beckman Coulter Pub. No. 987834

– Biomek® Software User’s Manual v4.1, Beckman Coulter Pub. No. B30026AA

• For information on recovering a run, contact your Thermo Fisher Scientific Field Application Scientist.

• For additional information on Biomek FXP Target Prep Express hardware, error messages, or to request service, contact Beckman Coulter. Be sure to have the serial number of your workstation available.

GeneTitan™ Multi-Channel Instrument

Refer to the GeneTitan™ Multi-Channel Instrument User Guide, Pub. No. 08-0306 for further troubleshooting information.

Table 13 GeneTitan Multi-Channel Instrument troubleshooting guidelines for the PharmacoScan Assay

Problem Possible causes Possible actions

Plate trapped in GeneTitan Multi-Channel Instrument.

• Plate (or plate with lid) not properly loaded in drawer.

• Notched edge of lid and plate not aligned.

• Gripper failed to retrieve plate.• System requires adjustment.

1. Restart the GeneTitan Multi-Channel Instrument.

2. Run the setup option Unload Plates.3. If the plate remains trapped in the

instrument, call Thermo Fisher Scientific support.

Computer frozen. • Too many processes running.• Attempting to transfer data while an

array plate is being scanned (imaged).

Restart the computer and unload all of the plates.• Plates in Hyb station: finish hybridization off-

line.• Plate in Scanner: rescan using Scan Only

function• Plate in Fluidics: use Wash/Scan Resume to

resume the fluidics process.Do not manually, or through the AGCC transfer utility, move any data associated with the current plate that is being processed/scanned.


Chapter 8 TroubleshootingGeneTitan™ Multi-Channel Instrument8

Miscellaneous messages

Hybridization aborted:• System-initiated abort• User-initiated abort

System-initiated abort:• Power lossUser-initiated abort:• User error• Other

Array plate and hyb tray are still clamped:• Contact your local Field Service Engineer

with information on the workstation model.• The plate stack is moved to drawer 1.• Remove the plate stack and finish

hybridization offline.• Return the hybridized array plate stack to the

GeneTitan Multi-Channel Instrument and finish processing using the Wash/Scan process.

FAILED messages See "GeneTitan MC Instrument messages that appear when the instrument has a fluidics problem" on page 218

FLUIDIC DIAGNOSTIC messages

See "Fluidic diagnostic messages" on page 218.

Fluidics aborted:• System-initiated abort• User-initiated abort

System-initiated abort:• Power lossUser-initiated abort:• Incorrect protocol selected

Follow the recommendations and instructions under "Wash/Scan Resume" on page 222.

Table 13 GeneTitan Multi-Channel Instrument troubleshooting guidelines for the PharmacoScan Assay

Problem Possible causes Possible actions

Table 14 Miscellaneous messages and recommended actions

Message and recommended action

Indicates that an item is in the gripper, and normal startup of the GeneTitan Multi-Channel Instrument is not possible. The item must be removed from the instrument before you can begin processing array plates.

Recommendation: click Yes.If you click No, nothing will occur. Homing will not complete and you will not be able to use the system.The item held by the gripper will be moved to either:• Drawer 2—plates and trays• Trash Bin—coversThe drawer names will reflect the location (left or right) and the drawer number (1 through 6).Examples: Drawer2L_Hta_DOWN = Scan tray on left side of drawer 2HtaHyb = Clamped Hyb Tray and Array PlateDrawer(n)L/R_Hta_DOWN where n is the drawer number and L or R to indicate the left or right side.The _Hta_ (second term) indicates the item held. An example is drawer1R_HtaHyb_DOWN indicating it is an array plate with a hyb tray or Drawer2L_ScanHta_Pk_DOWN indicating it is an array plate with a scan tray


Chapter 8 TroubleshootingGeneTitan™ Multi-Channel Instrument 8

The drawer listed in the message is not fully closed. Manually push the drawer back into the instrument until it is fully closed. There are two stop positions with audible clicks; push until you hear the second click and the drawer is fully seated.

• Check that the array plate barcode has been entered correctly.

• Ensure that the library files required for the type of array plate you are using have been installed, and are installed in the correct directory.

• Restart the GeneTitan Instrument control software after library files have been installed.

Table 14 Miscellaneous messages and recommended actions (Continued)

Message and recommended action



Fluidic diagnostic messages

Table 15 GeneTitan MC Instrument messages that appear when the instrument has a fluidics problem

Problem and possible causes

Rinse bottle—fluid level too low or bottle empty.

If this message is displayed:• during a water wash step, array processing has

been compromised.• during cleanup, array processing is okay, but

cleanup will not be complete.Always ensure that the GeneTitan bottles containing Wash A and Rinse are above the 50% mark when setting up the system to process an Axiom array plate.All 600 mL of the Wash Buffer B from the Axiom 2.0 reagent Kit should be emptied into the GeneTitan Wash B bottle when setting up the system to process a plate. This ensures that the GeneTitan Wash B bottle is filled to more than the requisite 35% of Wash B bottle volume.

About this message:• BUFFERX = Buffer bottle A, B or Rinse• WASHX = Wash A or B reservoir in the fluidics

station.Recommended actions:• Replenish fluid level in the Rinse or Wash Bottle B

to the 1L mark. Do not overfill.– Only replenish bottles when prompted by the UI.

Replenishing during fluidic processing may cause system malfunction including overflowing inside the system and more problems. The only thing to do while a plate is running is to make sure bottle caps are secure.

• Replenish fluid level in Wash Bottle A to 2L.• Secure the bottle cap.• Replace the filterInstructions for filter replacement in the GeneTitan™ Multi-Channel Instrument User Guide, Pub. No. 08-0308.If the problem persists, call Thermo Fisher Scientific Support.



The typical cause is an unsecure bottle cap.

If the failure is detected during priming, the instrument will pause and wait for the problem to be corrected.

If the failure is detected during another process, and if the cause is a clogged filter, wait until the end of the run to replace the filter.Instructions for filter replacement in the GeneTitan™ Multi-Channel Instrument User Guide, Pub. No. 08-0306.

When the instrument experiences a loss in Clean Dry Air (CDA) pressure, the software will display the warning message.

When the pressure is detected again, a dialog message confirming the availability of CDA pressure is displayed.

Possible causesPlease verify that the facility CDA or the portable CDA compressor is in working condition. Refer to the GeneTitan MC Instrument Site Preparation Guide for the portable compressor model that has been validated with the GeneTitan MC instrument.Contact your local Field Service Engineer and notify them about the error message.





Leak DetectedLeak checks are performed at application startup and any time a fluidic process (priming filling draining etc.) is performed. The leak detection is a hard-wired sensor which will shut off fluid flow without software control. Leaks are normally confined to the drip pan located inside the system.

Causes:• System malfunction• User killing the application using task manager

during a fill operation resulting in application exit without stopping flow.

Solution: Contact Support. The system cannot be used for any fluidic processing until this is resolved.

Leak Resolved This message is displayed when the leak is resolved (meaning the sensor LED is again lit up). If the original leak detected message was not acknowledged it will be automatically removed from the GUI and replaced by the following message. It will remain displayed until another leak is detected or the user acknowledges it by pressing OK. To resolve this issue complete the following tasks• Verify all internal and external tubing is connected

and clean• Verify wash reservoirs are clean• Verify all bottle caps are secure and that no bottle

cap is crimping a supply line.• Verify vacuum is working properly• Do not refill bottles or empty waste except when

prompted to by the GeneTitan application.• Contact your facility group to ensure CDA is

supplied to your GeneTitan system.Contact your Thermo Fisher Scientific Field Service Engineer to have the sensor adjusted or replaced if the problem persists even after correcting for the usual causes outlined above.





Filter Error Message: Dispense related check

Filter Error Message: Fill related check

The filters in the GeneTitan fluidics bottles (Wash A, Wash B and Rinse) need to be replaced when the filters are worn out. The software displays warning message boxes for the filter in each reagent bottle when it detects a problem or shows a trend of increased fill times during fluid fill operations. If an error is detected as described above, then a message box titled “Filter Change Required” is displayed along with the information on the specific dispense operation. You should change all three filters when a warning is displayed for any one of the three filters.Refer to the section "Replacing the filter" on page 248 in Appendix F.





Wash/Scan Resume

If a run is aborted during fluidics processing, the instrument will place the aborted array plate into the scan tray. To restart this process, remove the Axiom array plate from the scan tray and place the array in its protective blue base.

The step at which the run was aborted can be identified by:

• Viewing the System Status window if you are aborting the last plate through the fluidics system.

• Initiating the resume process.

1. System Setup tab: Select Wash/Scan Resume

2. Follow the prompts to unload and reload all drawers.

The trays will be loaded. It is up to you to determine whether or not to load fresh reagents or reuse the trays already in the GeneTitan Multi-Channel Instrument. Base your decision upon the step where the problem occurred.

To help ensure that the samples are processed correctly, we recommend that you:

1. Load new stain trays with fresh reagents.

2. Load a new scan tray.

We do not recommend the use of trays without reagents or holding buffers for steps that appear to have already executed.

Resume stepYou must select the step at which you wish to resume plate processing. You can select any step that has not yet been started.

For certain steps, you can enter a duration in seconds (even if the step requires > 1 hr to run, you must enter the duration in seconds). You can set a step for less time than normal, but not for longer than the normal duration.

Aborting a run • Abort can take up to three minutes if a plate is in the fluidics station. Status window Abort Requested changes to Abort Completed.

• Clamped Array-Plate-Hyb Tray stack that is aborted from the oven or from drawerIN (drawer 6) is moved to drawer 1.

• Proceed as follows:

– Use the Unload Plates option to remove the aborted plate(s).

– Start another run which will force an unload of the aborted plate(s)

System-initiated• Power interruption

• Plate loaded incorrectly

• Equipment malfunction

The system will abort the processing. Follow the instructions displayed in the user interface.

User-initiatedCan abort processing of individual array plates.

If multiple plates are being processed, the gripper may continue to process the remaining array plates.


A Safety

General safety

For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.

WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document.

• Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device.

• Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc). To obtain SDSs, see the ʺDocumentation and supportʺ section in this document.

WARNING! The following components contain harmful or toxic ingredients:

• Axiom Stabilize Soln: 8% Gluteraldehyde

• Axiom HybSoln 2: 100% Formamide

• Axiom Hyb Buffer: <55% Tetramethylammonium Chloride

In all cases customers should use adequate local and general ventilation in order to minimize airborne concentrations.


Appendix A SafetyChemical safetyA

Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, and consult the relevant SDS for specific precautions and instructions:

• Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the ʺDocumentation and supportʺ section in this document.

• Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing).

• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood).

• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturerʹs cleanup procedures as recommended in the SDS.

• Handle chemical wastes in a fume hood.

• Ensure use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.)

• After emptying a waste container, seal it with the cap provided.

• Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory.

• Ensure that the waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations.

• IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply.


Appendix A SafetyBiological hazard safety A

Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Safety equipment also may include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment.

• U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009; found at:www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

• World Health Organization, Laboratory Biosafety Manual, 3rd Edition, WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf


http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

B Fragmentation quality control gelprotocol

Protocol for running a fragmentation quality control gel

Equipment required

E-Gels and reagents

Consumables

Table 16 Equipment required


Gel imager Your choice —

Pipette, multi- or single-channel P20 Your choice —

Plate centrifuge Your choice —

Vortex Your choice —

Table 17 E-Gel and reagents required



Life Technologies(formerly

Invitrogen)

EB-M03



TrackIt 25 bp DNA Ladder 10488-022

TrackIt Cyan/Orange Loading Buffer 10482-028

Nuclease-free Water Your choice —

Table 18 Gel and reagents required


Adhesive film – use one of the following:• MicroAmp Clear Adhesive Film• Microseal 'B' Film

Thermo Fisher ScientificBio-Rad

4306311MSB1001

Pipette Tips Same brand as pipette —


Appendix B Fragmentation quality control gel protocolProtocol for running a fragmentation quality control gel B

Diluting the TrackIt™ Cyan/Orange Loading Buffer

The following recipe is for preparing a 1000-fold dilution of the TrackIt Cyan-Orange Loading Buffer.

To dilute the TrackIt Cyan/Orange Loading Buffer:

1. Add 50 µL of TrackIt Cyan/Orange Loading Buffer to 49.95 mL nuclease-free water.

Total volume 50 mL.

2. Vortex tube to mix well.

3. Store at room temperature.

To dilute the TrackIt 25bp Ladder:

The following recipe is for preparing a 15-fold dilution of the Invitrogen TrackIt 25 bp DNA Ladder.

1. In a 1.5 mL microcentrifuge tube, add 6 µL of TrackIt 25 bp Ladder to 84 µL nuclease-free water. Total volume: 90 µL.

2. Vortex tube to mix well. Pulse-spin to get droplets down.

Note: The recipe has enough volume to fill 4 marker wells of one E-Gel® 48 4% agarose gel. Scale up as needed if running multiple gels.

Fragmentation QC gel protocol

This protocol is based on running QC gels for 96 samples.

To run a fragmentation QC gel:

1. Tightly seal the gel QC plate prepared during automated target preparation.

2. Vortex the center of the plate for 3 sec. Pulse-spin to 1000 rpm to get droplets down.

3. Connect an E-Base™ device(s) to an electrical outlet.

4. Push the Power/Prg button on each to ensure the program is in EG mode (not EP mode).

5. Take the gel out of the pouch and remove the combs.

6. Place the E-Gel® 48 gel into an E-Base unit.

7. Load 20 µL from each well of the Gel QC plate onto the gels.

8. Load 15 µL of 15-fold diluted TrackIt 25 bp ladder into the marker wells (M).

9. Load 20 µL nuclease-free water into any unused wells.

10. Run the gels for 22 min.

11. Image the gel.

Fragmentation QC gel images should look similar to the gel shown in Figure 8.


Appendix B Fragmentation quality control gel protocolProtocol for running a fragmentation quality control gelB

Figure 8 Example of a typical fragmentation QC E-gel

125 bp

25 bp

25 bp

125 bp

Fragments should fall between 125 bp and 25 bp.

25 bp ladder 25 bp ladder


C Sample quantitation afterresuspension

Protocol for sample quantitation after resuspension

Equipment required The following equipment is required for this protocol.

Quantitate the diluted samples

During target prep, two plates of diluted samples are prepared: one for OD quantitation and one for a QC gel to check the fragmentation reaction.

For OD quantitation, readings should be taken at wavelengths of 260, 280, and 320 nm. See ʺSuggested protocol for OD quantitation using the DTX 880ʺ on page 231 for more information.

To quantitate the diluted samples prepared for OD quantitation:

1. Launch the Multimode Analysis Software.

2. When the Protocol Selection List is displayed, select the appropriate protocol.

3. Right-click the protocol and select Run the selected protocol.

4. In the Result Name field, enter your experiment name.

5. Click the Eject Plate Carrier icon.

6. Load the OD plate onto the DTX 880.

7. Click the Close Plate Carrier icon.

8. Click the Run the Selected Protocol icon at the bottom of the window.

When the protocol is finished running, a list of results is displayed. If you used the formula provided in this appendix, two XML files are generated (Figure 9). Open the ResultData file with Microsoft® Excel® to view and assess the OD readings. RawData file information is included in the ResultData file.

Table 19 Equipment required for sample quantitation after resuspension

Quantity Item

1 DTX 880 Multimode Detector with genomic filter slide

Figure 9 List of files that are generated post DTX-880 scan.


Appendix C Sample quantitation after resuspensionProtocol for sample quantitation after resuspensionC

Assess the OD readings

If using the formula provided in this appendix, the raw data is included in the final Result Data file. Figure 10 is an example of a Result Data file. Your OD readings should be similar to those displayed below.

OD yield assessment guidelinesThe measurement of the yield of DNA after resuspension of the pellets is an important QC checkpoint in the Axiom 2.0 Assay. If the median yield for the plate is < 1200 µg DNA per sample:

• Pause the protocol.

• Assess each of the steps performed to that point to determine the possible source of the low yields.

This DNA yield corresponds to an A260 value of approximately 0.59 and an A260-A320 value of approximately 0.50.

Figure 10 Example of Result data file with acceptable OD readings


Appendix C Sample quantitation after resuspensionSuggested protocol for OD quantitation using the DTX 880 C

Suggested protocol for OD quantitation using the DTX 880

The formula suggested below requires six passes. The settings and formula are shown below.

Protocol Type—Analysis

General Settings—Enter a name for the protocol


Appendix C Sample quantitation after resuspensionSuggested protocol for OD quantitation using the DTX 880C

Technique Type—Select Absorbance

Labware—x_Abs_Greiner 96 UV clear std (96 Microplate Format)



Layout Settings—as appropriate for 96-array format plates

Method Selection—add (+) the three formulas created on the Data Reduction Page to the Group 1 box.



Data Reduction Page—create the formulas required for scans at 260, 280 and 320This protocol consists of six passes. Click Add new Pass to create passes two through six, shown in these figures below.



1

k


Appendix C Sample quantitation after resuspensionIf performing sample quantitation on a plate reader other than the DTX880 C

Output Settings—select Export to Microsoft® Excel® and Show Result Viewer

Save the protocol.

If performing sample quantitation on a plate reader other than the DTX880

Your plate reader should be calibrated to ensure accurate readings.

The total yield in µg per well can be calculated as:

(A - C)*D*V*E/P

Where:

A = the observed OD260

C = the observed OD320 (an estimate of a blank reading)

D = 120 (the net dilution factor when preparing the OD Sample plate as described in the Automated Target Preparation Protocol)

V = 115 (the volume of the sample in µL after the resuspension step)

E = 0.05 (the extinction coefficient of duplex DNA at 260 nm)

P = the optical path length for the plate type and plate reader used.

If your plate reader does not record the OD320, the OD260 of a blank solution of water only should be used for the parameter “C” above.

The optical path length is dependent on the type of plate and spectrophotometer used. Check your manufacturerʹs recommendations for the path length for your instrument and plate type or for recommendations on how to measure this quantity. The SpectraMax Plus384, described as an alternative spectrophotometer in the PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP Site Preparation Guide, Pub. No. 703473, can employ an automated path length detection system. Consult this instrument’s manual for more information.

The resulting yield calculations can be compared against the typical yields shown in column H of Figure 10 on page 230 and against ʺOD yield assessment guidelinesʺ on page 230.


D Registering samples in GeneChip™

Command Console™

Creating a GeneTitan™ Array Plate Registration file

A GeneTitan Array Plate Registration file is a Microsoft® Excel® spreadsheet that includes information on the samples you are processing on a single array plate. This information includes the array plate format, the array plate barcode, and sample file names so that you can track the samples that are loaded onto a particular array plate.

The version of Microsoft Excel must be 1997-2000 (file extension is .xls; not .xlsx).

To create a GeneTitan Array Plate Registration file:

1. In AGCC Portal, open the Samples menu and select GeneTitan Array Plate Registration (Figure 11).

Figure 11 Selecting GeneTitan Array Plate Registration


Appendix D Registering samples in GeneChip™ Command Console™

Creating a GeneTitan™ Array Plate Registration file D

2. Select the array plate to be processed on the GeneTitan MC Instrument (Figure 12 on page 239).

a. Select the array plate type.

b. Click Download.

3. Complete the registration file as follows:

a. Click the Microsoft Excel box on the bottom bar of the monitor to open the Excel spreadsheet.

b. Enter a unique name for each sample (Sample File Name) and any additional information you would like to include (Figure 13).

c. Do one of the following:

• If you are ready to load the array plate onto the GeneTitan MC Instrument, scan the array plate barcode and proceed to the next step.

• If you are not ready to load the array plate onto the GeneTitan MC Instrument, proceed directly to the next step.

4. Save the file as follows:

a. Open File Save As.

b. Enter a name for the array plate registration file.

c. Click Save.

By default, the file is saved in the Affymetrix_Downloads folder.

Figure 12 Selecting the type of array plate to be processed

Figure 13 Entering sample information into an Array Plate Registration file


Appendix D Registering samples in GeneChip™ Command Console™

Creating a GeneTitan™ Array Plate Registration fileD

5. When ready to load the array plate onto the GeneTitan MC Instrument:

a. Click the Browse button, navigate to the file, and click Open.

b. Scan the array plate barcode if not already scanned.

c. Click the Upload button (Figure 14), wait for the information to load, then click the Save button located at the bottom of the next page that is displayed.

If the samples are successfully registered, the message in Figure 15 is displayed.

Figure 14 Uploading the Array Plate Registration file to AGCC

Figure 15 Array plate samples successfully registered


E Deionization procedure forGeneTitan™ trays and covers

We recommend the use of the Zerostat 3 Anti-Static Gun (Cat. No. 74-0014) to deionize GeneTitan™ MC Instrument stain tray trays and lids.

Deionize the inner surface of each tray and cover:

• The surface of the tray with the wells that will hold reagents.

• The surface of the cover that will face the reagents.

IMPORTANT! Except for the Axiom™ array plates, scan tray and the hybridization tray, you must deionize all GeneTitan stain trays, stain tray covers and scan tray cover using an anti-static gun. You must do this before you fill the trays with reagents and before you place the covers on the trays. Deionization removes the static electricity. The presence of static electricity on the underside of the cover can cause the gripper to lift the tray along with the tray cover and can result in an aborted run. See Figure 16, Figure 17 and Figure 18.

CAUTION! Do not deionize the scan tray or hybridization tray.

Figure 16 Scan tray with cover. Deionize only the cover.


Appendix E Deionization procedure for GeneTitan™ trays and coversDeionization procedureE

Deionization procedure

The following process provides guidance on how to use the anti-static gun on the stain and scan tray covers only. See Figure 18.

1. Treat the plate or lid as if it were divided into 6 sections (see Figure 18), and deionize as follows.

2. Place a Kimwipe on the benchtop.

3. Place the stain tray on a table top. Use the anti-static gun to aim at the center of each of the six sections on a 96-well tray and pull the trigger. Ensure that a stream of ionized particles settles on all wells of the stain tray to dissipate the static electricity. Squeeze and release the trigger slowly 3 times over each section (Squeeze for approximately two seconds and release for approximately two seconds).

4. Place the stain tray cover with the flat surface facing upward on the Kimwipe.

5. Aim the anti-static gun (Cat. No. 74-0014) approximately one-half inch away from the flat surface and pull the trigger. As you pull the trigger move the gun across the cover so that the stream of ionized particles settles on all areas of the cover and dissipates the static electricity. Squeeze and release the trigger slowly 3 times over each section (squeeze for approximately two seconds and release for approximately two seconds).

Figure 17 Stain tray with cover. Deionize the cover and the tray.

WARNING! The deionization steps 4 and 5 will damage the Axiom arrays on the plate. Before using the anti-static gun, ensure that the Axiom array plates remain in their protective pouch and placed away from the deionization area. You must place the scan tray and hybridization tray away from the area where you are performing deionization.


Appendix E Deionization procedure for GeneTitan™ trays and coversDeionization procedure E

6. Place the treated cover or tray on the Kimwipe and lift it up (see Figure 18).


• If the Kimwipe does not cling to the plastic, proceed with the protocol.

• If the Kimwipe still clings to the plastic, then perform steps 3 and 4 again. If it continues to cling to the plastic, test the device using the ion-indicator cap to confirm that the unit is still releasing ions. Otherwise, it may be time to replace the unit.

Figure 18 Removing the static charge from stain trays and lids.

Treat the inside surface of stain trays (right) and cover (left).

• If a Kimwipe clings to treated surface, try the deionization procedure again. • If the Kimwipe still clings, it may be time to replace the anti-static gun.

1 2

3 4

5 6


Appendix E Deionization procedure for GeneTitan™ trays and coversIon-indicator capE

Ion-indicator cap

The ion-indicator cap is a testing device used to verify the release of ions when the anti-static gun is in use (Cat. No. 74-0014, Figure 18).

Testing the Zerostat 3 with the ion-indicator cap

1. Insert the ion-indicator cap into the nose of the Zerostat and then slowly squeeze the release trigger (see Figure 19).

2. Observe the discharge through the viewing slot on the ion-indicator cap of the anti-static gun. A visible light is observed in the viewing window on the cap when charged ions are discharged.

3. If you cannot see the light, the gun may be unusable and you should replace it.

4. Each Zerostat anti-static gun is capable of 50,000 trigger operations, which is sufficient for approximately 200-250 runs on the GeneTitan MC Instrument.

Figure 19 Zerostat 3 anti-static gun (Cat. No. 74-0014) with ion-indicator cap to test functionality

IMPORTANT! Make sure to remove the cap from the gun before deionizing a tray or cover.

Ion-indicator cap

The Ion-Indicator Cap is attached to the Zerostat to test the functionality of the anti-static gun.

IMPORTANT: Do not leave the Ion-Indicator Cap on the Zerostat gun when deionizing trays and lids.


F GeneTitan™ Multi-ChannelInstrument Care

Cleaning and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

Servicing the outer enclosure fan filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

Replacing the bottle filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

Replacing the xenon lamp in the GeneTitan™ MC Instrument . . . . . . . . . . . . . 249

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

This chapter provides instructions on caring for and maintaining the instrument and on troubleshooting if problems arise.

• Always run a Shutdown protocol when the instrument will be off or unused overnight or longer. This will prevent salt crystals from forming within the Fluidics system.

• Always use deionized water to prevent contamination of the lines. Swap out old buffers with freshly prepared buffer at each system startup.

The GeneTitan™ Instrument should be positioned on a sturdy level bench away from extremes in temperature and away from moving air.

Cleaning and maintenance

The GeneTitan family of instruments require little in the way of customer maintenance. The instruments must be kept clean and free of dust. Dust buildup can degrade performance. Wipe the exterior surfaces clean using a mild dish detergent solution in water. Do not use ammonia based cleaners or organic solvents such as alcohol or acetone to clean the system because they may damage the exterior surfaces.

The following tasks should be performed regularly to ensure the Imaging Device remains in working order.

Monthly Wipe down the outer surface of the Imaging Device with a dry cloth.

Every six months Replace the cooling fan air filters at the rear of the instrument.

Replace the Micropore filters in the Wash A, Wash B, and Rinse bottles. If you run 4-8 plates/week then the micro-pore filters need to be replaced more frequently.

IMPORTANT! Before performing maintenance turn off power to the instrument to avoid injury in case of an electrical malfunction.


Appendix F GeneTitan™ Multi-Channel Instrument CareServicing the outer enclosure fan filtersF

Servicing the outer enclosure fan filters

Cleaning schedule The GeneTitan fan filter cartridge (Figure 20) should be cleaned at least every 90 days of service. Note that in some service locations, the presence of excessive dust or particulate matter may necessitate cleaning the cartridge more often than 90 days.

A plugged filter cartridge can cause excessive temperatures within the machine that can cause unwanted evaporation of GeneTitan reagents.

Part details for GeneTitan fan filter:Thermo Fisher Scientific Cat. No. 01-0669

Number of filters required per GeneTitan instrument: 3

Cleaning procedure 1. Slide the filter cartridge from the fan filter cartridge at the rear of the GeneTitan MC Instrument.

2. Submerse in clean DI water. Rinse and agitate gently to dislodge material.

3. Remove from water and dry with clean compressed air or towels.

4. When the filter cartridge is completely dry to the touch, re-install the cartridge.

Figure 20 The GeneTitan filter cartridge


Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the bottle filters F

Replacing the bottle filters

The bottles used in GeneTitan MC Instrument contain a filter to remove particulates that may exist in the buffers and DI water. The filters in the GeneTitan fluidics bottles (Wash A, Wash B and Rinse) need to be replaced when the filters are clogged.

The message boxes displayed in Figure 21 will provide information on fluid dispense errors that were detected by the instrument for any of the bottles or when the instrument detects an increase in the amount of time that is required to perform the fill operations.

If an error is detected as described above, then a message box titled “Filter Change Required” is displayed (Figure 21) along with the information on the specific dispense operation. You should change all three filters when a warning is displayed for any one of the three filters.

Note: The reagent bottles are depressurized when this warning message is displayed. It is safe to change the filters in all three fluidic bottles when this message is displayed.

After changing the filters in all three bottles using the procedure described below, please press the Yes button to continue. If you choose to ignore the error message, press the No button. This warning message will be displayed each time AGCC instrument control software is launched. You may also experience data quality issues if particulate matter cannot be trapped by the filters because they are clogged.

Figure 21 Filter Change Required messages


Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the bottle filtersF

We recommend that your site keep three spare filters on hand in the event the filters need to be replaced. The procedure for replacing the filters is simple.

GeneTitan reagent bottle filters part details:Thermo Fisher Scientific Cat. No. 01-0671

Removing and inspecting the filter

1. Loosen and remove the cap on the bottle.

2. Carefully remove the filter from the end of the filter body.

3. Visually inspect the filter. If one of the filters appears to have a concentration of dirt or contaminate in it, discard it and replace the filter with a new one.

Replacing the filter 1. Insert the filter into the end of the filter body.

2. Replace the cap onto the bottle and tighten it.

3. Repeat for each bottle.

Figure 22 Replacing the filter

Buffer supply line

Filter holder

Filter

1

2

3

1 2 3

IMPORTANT! Replace one filter at a time to ensure the correct connection of the buffer supply tube to its respective bottle. The color of the buffer supply tubing matches the bottle color code.


Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the xenon lamp in the GeneTitan™ MC Instrument F

Replacing the xenon lamp in the GeneTitan™ MC Instrument

This section applies to your site only if you have the GeneTitan Multi-Channel (MC) instrument. After the normal life expectancy of the lamp has expired, the software application will alert you to the requirement to replace the lamp. This procedure is simple but you must follow good health and safety precautions.

GeneTitan xenon lamp catalog number: Thermo Fisher Scientific 01-0740

Lamp life/imaging device status notices

The Imaging Status pane displays lamp life and Imaging Device status notices for the GeneTitan MC Instrument.

In normal operation, the pane displays the hours of life left in the lamp (Figure 23):

It displays a red or yellow notice when the lamp life is getting short (Figure 24):

It also displays a red notice when the Imaging Device is offline (Figure 25):

Note: The 300 watt xenon lamp in the GeneTitan MC Instrument is warranted for 500 hours. The instructions to replace the lamp are available on the following page. After changing the lamp, it is necessary to reset the lamp life clock manually.

IMPORTANT! Please DO NOT try to replace the lamp when a plate is being processed either in the fluidics or scanner system.

Figure 23 Lamp Life above tolerance

Figure 24 Lamp Life above tolerance

Figure 25 Imaging Device offline

WARNING! You must turn off the lamp using the power switch in the rear of the unit and remove the power cord. Allow the lamp to cool before attempting to replace the lamp.


Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the xenon lamp in the GeneTitan™ MC InstrumentF

Removing the xenon lamp

1. Unscrew the four retaining bolts. They should be finger tight (Figure 26).

2. Remove and set aside the warning cover to reveal the xeon lamp contained within.

3. Place each hand on each side of the blue plastic flange and lift out the lamp in a vertical motion (Figure 27). You must use both hands to remove the lamp successfully. Apply equal pressure on each side of the lamp and gently lift.

Figure 26 Unscrewing the Bolts

Figure 27 Lifting out the lamp

Unscrew these four bolts.



Replacing the lamp

1. Hold the lamp by the blue plastic flanges. Ensure that the lamp bulb faces inward toward the reflecting mirror (Figure 28) and vertically insert the lamp (Figure 29).

2. Replace the warning cover and hand tighten the bolts (Figure 26).

CAUTION! Ensure that you install the lamp in the correct orientation.

Figure 28 The reflecting mirror

Reflecting mirror


Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the xenon lamp in the GeneTitan™ MC InstrumentF

Figure 29 Inserting the lamp

IMPORTANT: The lamp bulb faces away from the fan and toward the reflecting mirror.



Resetting the lamp counter

You must alert the software application that you have replaced the lamp so that the hours of the lamp counter are reset to zero. This menu option is only available when the system is not processing any plates.

1. On the software application click Tools Reset Counter for Life Remaining (Figure 30).

2. The software will display a message that asks you to confirm the lamp life counter is being reset as a result of lamp replacement (Figure 31).

Figure 30 Inserting the lamp


Appendix F GeneTitan™ Multi-Channel Instrument CareTroubleshootingF

3. Click Yes if you want to reset the counter. The software will display a message that confirms that the software has reset the counter (Figure 32).

Troubleshooting

This section provides instructions on how to identify and solve simple problems with the GeneTitan MC Instrument. If a problem or error occurs that is not listed in this chapter contact Thermo Fisher Scientific Technical Support for assistance.

For software errors that do not involve hardware crashes the most common solution is to shut down the application and then restart it. If the same error occurs shut down both the application and the computer and then restart. If it still occurs shut down the GeneTitan MC Instrument and then restart.

Log files The log files are produced by different AGCC components. The logs provide a record of the tasks performed by different components, such as the migration tools and installer. These log files provide useful information for troubleshooting problems. These files may be requested by your Field Application Scientist (FAS), Field Service Engineer (FSE), or the call center.

Figure 31 Are you sure?

Figure 32 The counter is reset.


Appendix F GeneTitan™ Multi-Channel Instrument CareTroubleshooting F

AGCC log filesThe following files are generated by the GeneTitan Instruments. All the AGCC log files are from the following path: C:\Command_Console\Logs. The different log files include:

Other AGCC filesYour FAS and/or FSE may request you to send the following files for troubleshooting:

1. Library files (*.PARAMS, *.MASTER, *.WORKFLOW, *.SMD, *.MEDIA) located in C:\Command_Console\Library, excluding the large analysis library files (CDF, PSI, GRC).

2. Provide a list of all sub folders and their contents under the library files folder located in C:\Command_Console\Library. Please ensure there are no duplicate library files, as these can cause problems.

3. AGCC system configuration file located at C:\Command_Console\Configuration\Calvin.System.config

4. Pending job order files located in C:\Command_Console\Jobs

5. Other AGCC related information, such as:

a. The number of files under C:\Command_Console\Data, including sub directory.

b. If the system is a networked system or a standalone system.

c. Other applications installed on the system, such as antivirus application, MS Office, and Internet Explorer versions.

AGCC log files for GeneTitan™ MC Instrument Systems

Log files for the GeneTitan MC Instrument control processes are placed in subdirectories of the C:\Command_Console\Logs\ folder. Thermo Fisher Scientific may need the following files for troubleshooting:

GeneTitan MC Instrument fluidics

1. C:\Command_Console\Logs\96F\

a. Subdirectories named by date (e.g., Log7-29-2009)

• Collect all dated directories and contents since the GeneTitan application was started, not just the date of the event (some logging goes into files from the date the application started so this can be critical for us).

• Absolutely required are all the log directories from the date the run was started to the date of the event.

2. C:\Command_Console\Logs\96F\FluidicErrorLog - all files in this directory.

Systemlog.XML XML file with system information.

DEC.log Text file with information on the use of the Data Exchange Console (DEC).

DECError.log Text file with information on errors created while using DEC.

AGCC_LibFileImporter. log (with date and time code)

Text file with info on use of the Library File Importer.


Appendix F GeneTitan™ Multi-Channel Instrument CareTroubleshootingF

GeneTitan MC Instrument imaging device

1. C:\Affymetrix\GeneChipHTScanControlMC\Log - collect all dated directories and contents since the GeneTitan application was started.

2. C:\Affymetrix\GeneChipHTScanControlMC\RunLog - collect all dated directories and contents since the GeneTitan application was started.

Insufficient disk space notice

If there is not enough memory on the computer’s drives to save the data from an array plate, a notice appears (Figure 33) when:

• you first initialize the software and instrument.

• you select arrays for imaging.

If you see this notice, you will need to free up sufficient disk space before imaging starts.

Figure 33 Insufficient disk space notice


G mPCR quality control gel protocol

Protocol for running an mPCR quality control gel

Note: This is an optional procedure. It is meant only as a QUALITATIVE examination of the mPCR reaction to confirm that amplification has occurred. Gene copy number differences will result in differences in DNA band patterns and amplicon intensities, and therefore sample to sample variation maybe observed.

Equipment required

E-Gels and reagents

Table 20 Equipment required

Item Supplier Cat. No.

Gel imager Various —

Pipette, multi- and single-channel Various —

Plate centrifuge Various —

Vortex Various —

Table 21 E-Gel and reagents required



Thermo Fisher Scientific

EB-M03



TrackIt™ Cyan/Orange Loading Buffer 10482-028

Reduced EDTA TE Buffer (10 mM Tris-HCL PH 8.0, 0.1 mM EDTA)

75793

NEB 50 bp DNA Ladder New England BioLabs N3236S

Diluted TrackIt Cyan/Orange Loading Buffer Refer to "Diluting the TrackIt™ Cyan/Orange Loading Buffer" on page 227


Appendix G mPCR quality control gel protocolProtocol for running an mPCR quality control gelG

Consumables

Prepare NEB 50 bp DNA ladder

Dilute the NEB 50 bp ladder (Cat. No. N3236S, New England BioLabs):

The following recipe is for preparing a 250-fold dilution of the NEB 50 bp DNA Ladder (4 ng/µL final concentration):

1. In a 1.7 mL microcentrifuge tube, add 1 µL of 50 bp DNA Ladder to 249 µL of 1000-fold diluted TrackIt dye.

2. Vortex tube to mix well. Pulse-spin to get droplets down.

Preparing mPCR samples for gel analysis

1. Thaw mPCR Reaction plate on benchtop at room temperature.

2. Ensure plate seal is secure, vortex plate, and pulse spin.

3. Dilute mPCR reaction samples 120-fold by:

a. First dilute mPCR samples 12-fold in buffer. Transfer 2 µL of the mPCR reaction into 22 µL of low EDTA TE. Seal plate. Vortex. Pulse-spin.

b. Then dilute samples another 10-fold in loading dye to prepare “mPCR Gel QC Plate”. Transfer 2 µL of the 12-fold diluted mPCR reactions into 18 µL of 1000-fold diluted TrackIt dye.

mPCR QC gel protocol

This protocol is based on running QC gels for 96 samples. Two E-Gel® 48 2% agarose gels will be needed.

To run mPCR QC gel:

1. Tightly seal the mPCR Gel QC plate.

2. Vortex the plate for 3 sec. Pulse-spin to get droplets down.

3. Connect an E-Base™ device(s) to an electrical outlet.

4. Push the Power/Prg button on each to ensure the program is in EG mode (not EP mode).

5. Take the gel out of the pouch and remove the combs.

6. Place the E-Gel 48 gel into an E-Base unit.

7. Load 15 µL from each well of the mPCR Gel QC plate onto the gels.

8. Load 15 µL of diluted 50 bp ladder into the marker wells (M).

9. Load 15 µL nuclease-free water into any unused wells.

10. Run the gels for 25 min.

11. Image the gel.

Table 22 Consumables Required


Adhesive film – use one of the following:• MicroAmp Clear Adhesive Film• Microseal 'B' Film

Thermo Fisher Scientific Bio-Rad

4306311MSB1001

Pipette Tips Same brand as pipette —

96-well PCR plate Various Various

1.7 mL microcentrifuge tube Various Various


Appendix G mPCR quality control gel protocolProtocol for running an mPCR quality control gel G

mPCR QC Gel images should look similar to the gel shown in Figure 34.

Note: Variation in DNA band patterns and intensities maybe observed from sample to sample due to gene copy number differences. The mPCR QC Gel is meant to be a qualitative examination of the mPCR reaction to confirm that amplification has occurred for each sample.

All samples were amplified and show DNA bands that fall between 150 bp and 700 bp.

Figure 34 Example of a typical mPCR QC E-gel

50 bp ladder

700 bp

50 bp ladder

150 bp


Documentation and support

Related documentation

Table 23 Documents related to the PharmacoScan™ Assay 96-Array Format Automated Protocol for Biomek FXP

Document Publication number Description

PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP (Windows® 7) Site Preparation Guide

703473 Provides guidance on reagents, instruments, and supplies required to run the PharmacoScan Assay 96-Array Format Automated Protocol for Beckman Biomek FXP.

PharmacoScan™ Assay 96-Array Format Automated Protocol for Beckman Biomek FXP (Windows® 7) Quick Reference

703474 An abbreviated reference for the target preparation step of the PharmacoScan Assay 96-Array Format Automated Protocol for Biomek FXP running on the Windows 7 operating system. This quick reference document is intended for experienced users.

Axiom™ 2.0 gDNA Sample Prep Protocol QR

702987 An abbreviated reference on the genomic DNA sample preparation protocol.

Axiom™ gDNA Sample Prep for Genome-Wide BOS 1 Array Plate QR

702975 An abbreviated reference on the genomic DNA sample preparation protocol for the Genome-Wide BOS 1 Array Plate.

GeneTitan™ MC Protocol for Axiom™ 2.0 Array Plate Processing QR

702988 An abbreviated reference for processing Axiom 2.0 array plates with the GeneTitan Multi-Channel Instrument.

GeneTitan™ Multi-Channel Instrument User Guide

08-0308 The GeneTitan Multi-Channel (MC) Instrument automates array processing from target hybridization to data generation by combining a hybridization oven, fluidics processing, and state-of-the art imaging device into a single bench-top instrument. This document detailing the use, care, and maintenance for the GeneTitan MC Instrument.

GeneTitan™ Multi-Channel Instrument Site Preparation Guide

08-0305 Provides guidance on creating and maintaining the proper environment required for the GeneTitan Multi-Channel Instrument.


Documentation and supportRelated documentation

Analysis and Software

Axiom™ Genotyping Solution Data Analysis Guide

702961 This guide provides information and instructions for analyzing Axiom genotyping array data. It includes the use of Axiom™ Analysis Suite, Applied Biosystems Microarray Power Tools (formerly APT) and SNPolisher R package to perform quality control analysis (QC) for samples and plates, SNP filtering prior to downstream analysis, and advanced genotyping methods.

Applied Biosystems ™ GeneChip™ Command Console™ Software User Guide

702569 This user guide provides instructions on using Applied Biosystems GeneChip Command Console Software (AGCC) used to control GeneChip instrument systems. Command Console Software provides an intuitive set of tools for instrument control and data management used in the processing of GeneChip Arrays.

Axiom™ Analysis Suite User Guide 703307 This user guide provides instructions on using Axiom™ Analysis Suite—a single-source software package to enable complete genotyping analysis of all Axiom arrays.

PharmacoScan Assay Manual Protocol

PharmacoScan™ Assay 96-Array Format Manual Protocol Site Preparation Guide

703459 This user guide provides comprehensive instructions on running the PharmacoScan Assay 96-Array Format Manual Protocol


703460 Provides guidance on reagents, instruments, and supplies required to run the PharmacoScan Assay 96-Array Format Manual Protocol.

PharmacoScan™ Assay 96-Array Format Manual Protocol Quick Reference

703461 An abbreviated reference for the target preparation step of the PharmacoScan Assay 96-Array Format Manual Protocol. This quick reference document is intended for experienced users.

PharmacoScan™ Assay 24-Array Format Manual Protocol User Guide

703286 This user guide provides comprehensive instructions on running the PharmacoScan Assay 24-Array Format Manual Protocol.


703287 Provides guidance on reagents, instruments, and supplies required to run the PharmacoScan Assay 24-Array Format Manual Protocol.

PharmacoScan™ Assay 24-Array Format Manual Protocol Quick Reference

703288 An abbreviated reference for the target preparation step of the PharmacoScan Assay 24-Array Format Manual Protocol. This quick reference document is intended for experienced users.




Documentation and supportRelated documentation

Beckman Coulter documents

Biomek® Liquid Handler User’s Manual 987834 This document is installed at the same time as the Biomek FXP Target Prep Express software. To access, click Start All Programs Beckman Coulter Manuals.

Biomek® Software User’s Manual B30026AA This document is installed at the same time as the Biomek FXP Target Prep Express software. To access, click Start All Programs Beckman Coulter Manuals.




Documentation and supportCustomer and technical support

Customer and technical support

Visit thermofisher.com/support for the latest in services and support, including:

• Worldwide contact telephone numbers

• Product support, including:

– Product FAQs

– Software, patches, and updates

• Order and web support

• Product documentation, including:

– User guides, manuals, and protocols

– Certificates of Analysis

– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies’ General Terms and Conditions of Sale found on Life Technologies’ website at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at thermofisher.com/support.


http://thermofisher.com/support

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html


References

Hindorff LA, Junkins HA, Mehta JP, and Manolio TA. A Catalog of Published Genome-Wide Association Studies. Available at: www.genome.gov/gwastudies. Accessed 09/28/2009.

Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science 2005, 308:385–89

Manolio T.A. and Collins F.S. The HapMap and Genome-Wide Association Studies in Diagnosis and Therapy. Annu Rev Medicine 2009, 60:443–56


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16 May 2017


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