Kit for the extraction of fragment DNA from agarose gel using MagListo™

Version No.: 2.0 (2017-03)

Please read all the information in booklet before using the unit

Bioneer Corporation

8-11, Munpyeongseo-ro, Daedeok-gu, Daejeon

34302, Republic of Korea

Tel: +82-42-930-8777

Fax: +82-42-930-8688

Email: order@bioneer.co.kr

www.bioneer.com

MagListo™ is a trademark of Bioneer Corporation.

Copyright 2017. Bioneer Corporation. All Rights Reserved.

Contents

I. Overview 1

II. Kit Components 1

III. Storage 2

IV. Intended Use 2

V. Safety Warnings and Precautions 2

VI. Warranty and Liability 2

VII. Technical Assistance 3

VIII. Quality Management 3

IX. Kit Specifications 4

X. Principle 4

XI. Magnetic Nano Bead Information 5

XII. Guidelines for MagListo™ Magnetic Separation Rack 5

XIll. Materials and Equipment Needed But Not Provided 6

Types of the Magnetic Separation Rack 6

XlV. Procedure 7

XV. Protocol 8

Fragment DNA Extraction from Gel 8

Summary of Reagents Volume in Each Step 11

XVI. Appendix 12

Troubleshooting guide 12

Experimental data 14

XVII. Ordering Information 15

XVIII. Explanation of Symbols 16

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I. Overview

Description

MagListo™ 5M Gel Extraction Kit utilizes Magnetic Nano Beads to extract up to 10 μg of fragment DNA

from agarose gel with 1X TAE or TBE buffer within 15 min, with the aid of the MagListo™ Magnetic

Separation Rack. The use of MagListo™ Magnetic Separation Rack along with the kit greatly increases user

convenience by shortening the extraction time without centrifugation. The maximum amount of gel slice

per each sample is about 400mg. The size range for effective extraction about 100 bp to 10 Kb and the

average recovery yield exceeds 90% to 100%.

Features and Benefits

- Magnetic Nano Beads enable rapid extraction of fragment DNA

- No requirement of expensive instruments

Applications

Sub-cloning, Sequencing, Labeling, DNA concentration and other molecular biological applications

II. Kit Components

MagListo™ 5M Gel Extraction Kit K-3607

(100 reaction)

Buffer ① (Gel Solubilization) 60 ml x 2 ea

Buffer ② (1st Washing) 80 ml x 1 ea

Buffer ③ (2nd

Washing) 80 ml x 1 ea

Buffer ④ (3rd Washing) 80 ml x 1 ea

Buffer ⑤ (Elution) 15 ml x 1 ea

Magnetic Nano Bead - DNA 1.8 ml x 6 ea

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III. Storage

MagListo™ 5M Gel Extraction Kit should be stored dry at room temperature. It can be stored for up to 2

years if it remains sealed.

IV. Intended Use

MagListo™ 5M Gel Extraction Kit is intended for research use only. This kit is not intended for human or

veterinary diagnostics.

V. Safety Warnings and Precautions

The Buffer ⓛ (Gel solubilization) should be stored in a dark place and redissolved before use. The Buffer

① in the MagListo™ 5M Gel Extraction Kit is poisonous and hazardous. Please handle with care while

working with Buffer ① by wearing gloves and goggle eye protection.

Please inquire BIONEER’s Customer Service Center to obtain a copy of the Material Safety Data Sheet

(MSDS) for this product.

Before, during and after using this kit, all potentially hazardous materials (i.e. materials that may have

come in contacted with genetically recombinant samples) including tubes, tips and other kit contents

should be processed and discarded in accordance with applicable and appropriate regulations of the

municipality/government in which this product is being used. A user must also be equipped with basic

experimental techniques required for correct execution of the extraction experiments described in this

User’s Guide.

Some inventions applied in this kit may infringe existing patents in certain countries. The purchase of this

kit does not include or provide a license to perform such patented inventions. Users may be required to

obtain a license depending on the patent law of the country where this product is being used. We do not

condone nor recommend the unlicensed use of patented inventions.

VI. Warranty and Liability

All BIONEER products are manufactured and tested under strict quality control protocols. BIONEER

guarantees the quality of all directly manufactured products during the warranty period of one (1) year from

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the date of purchase. If you find any issues regarding the product quality, please immediately contact

BIONEER’s Customer Service Center (sales@bioneer.com).

BIONEER does not assume any liability for the misuse of the product, i.e. using the product for any

purposes other than its intended purpose as described in the User’s Guide. BIONEER will only assume

liability under the condition when the users disclose all related information regarding the issue to BIONEER

in written form within 30 days after occurrence of the issue.

VII. Technical Assistance

At Bioneer, we pride ourselves on being responsive to your needs. Our Technical Service Departments are

staffed by experienced scientists with extensive practical and theoretical expertise in Molecular Biology and

the use of Bioneer products. If you have any questions or would like to find out more information about

MagListo™ products, please contact us. We look forward to hearing from you!

Technical Support

For all technical questions and troubleshooting on Bioneer products and applications.

Tel: +82-42-930-8777

Email: sales@bioneer.co.kr

In North America

Tel: +1-877-264-4300

Email:support@bioneer.us.com

VIII. Quality Management

Every aspect of our quality management system from product development to supplier qualification

ensures that our products meet the world-class standards. Each lot of MagListo™ 5M Gel Extraction Kit is

carefully tested by the quality control team.

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IX. Kit Specifications

Gel slice ~ 400 mg

Size range 100 bp - 10 kb

Binding capacity ~ 10 g

Typical recovery 90%-100%

Expected purity A260/280 > 1.8

Purification time < 15 min

X. Principle

MagListo™ 5M Gel Extraction Kit is designed for the extraction of highly purified fragment DNA from

agarose gel with 1X TAE or TBE buffer. The overall principle is based on adsorption of DNA onto the

Magnetic Nano Bead by chaotropic and other salt components which also enhance melting of agarose gel.

For example, chaotropic agents in Buffer ⓛ (Gel Solubilization) contains such as guanidine thiocyanate,

as which removes water molecules around DNA and silica coated magnetic beads surface resulting in

fragment DNA then being captured by magnetic beads. The Magnetic Nano Bead and nucleic acid

complexes are pulled and fixed on the tube wall using a magnetic force, followed by washing with ethanol

to remove debris and excessive salts. Finally, the captured nucleic acids are then eluted by Buffer ④

(Elution), an aqueous solution with optimal pH.

Gel Solubilization Binding Washing Elution

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XI. Magnetic Nano Bead Information

Description

Magnetic Nano Beads have been developed to overcome shortcomings of existing resin and to automate

purification process. The principle of extraction using the Magnetic Nano Beads is that coated functional

group on the surface of the Magnetic Nano Beads bind DNA and the Magnetic Nano Beads are then

isolated using external magnetic field.

Features

Fast binding guarantees higher throughput automation

Large surface area enables more sensitive assay

Globular structure increases specificity by decreasing non-specific binding

Specification

XII. Guidelines for MagListo™ Magnetic Separation Rack

Description

MagListo™ Magnetic Separation Rack is designed for a fast, easy separation of the Magnetic

NanoBeads. These racks of different sizes allow users to choose the product according to their needs.

The following are recommended when handling the MagListo™ Magnetic Separation Rack

The product is made of acryl and plastic. Be careful not to drop the product as the dropping may

break the product.

When moving the product, take extra care not to drop the product as it may cause injury.

If the product is broken, do not discard it with bare hands as the sharp edges may cause injury.

AccuNanoBead™ Silica Magnetic Nano Beads

Matrix Silica-coated Fe3O4

Average size 400nm

Ligand -OH

Working Temp. 0-100℃

Storage Store at room temperature upon receipt

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When an extracted or purified nucleic acid is spilled on the product, immediately rinse it with running

water and clean it with 70% ethanol.

Acetone, Toluene, or organic solvent may damage the acrylic and plastic part of the product, which

may lead to malfunction of the product. Rinse the product immediately when spillage of any above

mentioned solvents occurs as the expected DNA yield may not be obtained if the product is

damaged.

Make sure that do not spill a corrosive liquid on the magnet plate part of the product. If spillage

occurs, immediately rinse it off with running water as it may corrode the magnet during storage and

may degrade its performance.

XIll. Materials and Equipment Needed But Not Provided

1. UV light-box

2. Scalpel

3. 1.5 ml tube or 2 ml tube

4. Vortex mixer

5. Absolute ethanol

6. Thermal block

7. MagListo™ Magnetic Separation Rack

Types of the Magnetic Separation Rack

Tube MagListo™ Magnetic Separation Rack

1 ml tube with 8-cap strip MagListo™-8Ch Magnetic Separation Rack

(Cat. no. TM-1000)

1.5 ml or 2 ml microcentrifuge tube MagListo™-2 Magnetic Separation Rack

(Cat. no. TM-1010)

(Note) Please refer to the ordering information in this User’s Guide for more information

regarding catalog number of racks designed for specific size of tubes.

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XIII. Procedure-Gel Extraction

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XV. Protocol

A. Fragment DNA Extraction from Gel

1. (Gel Cutting) Visualize the band, in an ethidium bromide stained gel, in a dark room with an UV light.

Cut around the band of interest DNA using a scalpel blade. Switch off the UV light-box, carefully

remove the slice from the gel and weigh the gel slice in a 1.5 ml or 2 ml tube (not provided).

(Note) Set the UV light-box to long wave length UV, if possible, and minimize the exposure time to DNA.

2. (Gel Solubilization: 2-3) The maximum amount of gel slice per each sample is ~ 400 mg. Add 3

volumes of Buffer ① (Gel Solubilization) to 1 volume of the gel slice. (If the weight of gel slice is 200

mg, add 600 μl of Buffer ①.)

3. Incubate at 60℃ for 10 min. Mix by inverting the tube every 2-3 min during the incubation. If the gel

slice is dissolved incompletely, increase the incubation time. After dissolving the gel slice, check the

color of the mixture whether it is yellow or not.

(Note) The color of the mixture indicates pH of the mixture related with DNA binding. pH ≤ 7.5 (yellow

color), the fragment DNA can effectively bind to the magnetic nano beads.

4. (DNA Binding with Magnetic Nano Bead: 4-6) Add 100 μl of Magnetic Nano Bead solution to the each

tube and mix thoroughly using a vortex mixer until the beads are fully resuspended.

(Note) Magnetic Nano Bead solution contains Magnetic Nano Beads. Please shake well or mix with a

vortex mixer before use.

5. Place the tubes on the MagListo™ -2 Magnetic Separation Rack with the magnet plate attached and

invert the rack gently 3 to 4 times until the beads bind tightly to the magnet.

- Attachment of the magnet plate

Combine the magnet plate to the stand.

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6. Without removing the tubes from the MagListo™ Magnetic Separation Rack, discard the supernatant

carefully and completely remove the remaining supernatant on a paper towel by blotting action. This

process, the magnetic crude pellet remains attached to the side of tube.

- How to discard the supernatant

Discard the supernatant by inverting the MagListo™ Magnetic Separation Rack. The silicone

immobilizer inside the stand prevents the tubes from. When discarding the supernatant, invert the rack

completely so that the solution odes not to spill on the rack.

7. (1st Washing: 7-9) Detach the magnet plate from the MagListo™ Magnetic Separation Rack. Add 700

μl of Buffer ② (1st Washing) to the each tube and close the cap. Mix using a vortex mixer until the

beads are fully resuspended.

- Detachment of the magnet plate

Detach the magnet plate gently by pulling it upwards.

8. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the

magnet.

9. Without removing the tubes from the MagListo™ Magnetic Separation Rack, discard the supernatant

and completely remove the remaining supernatant on a paper towel by blotting action.

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10. (2nd

Washing) Repeat the steps 7 - 9 by adding 700 μl of Buffer ③ (2nd

Washing) instead Buffer ②

for additional washing.

11. (3rd Washing) Without removing the tubes from the MagListo™

Magnetic Separation Rack, add 700 μl

of Buffer ④ (3rd Washing) to “the opposite side of bead pellet” close the cap and invert the rack twice

in order to remove ethanol from the sample.

(Note) Direct pipetting of Buffer ④ onto the bead pellet, vortexing and/or vigorous shaking of the

tubes may release nucleic acid from the beads, which may result in lower DNA yield than expected

12. Discard the supernatant and completely remove the remaining supernatant by blotting action.

- Add Buffer ④ and discard the supernatant

13. (Elution: 13-17) Detach the magnet plate from the MagListo™ Magnetic Separation Rack. Add 30 ~

50 μl of Buffer ⑤ (Elution) or distilled water to the each tube and resuspend the bead pellet

completely by pipetting or using a vortex mixer for 15 sec.

14. Incubate the tube at 60℃ for 1 min.

15. Vortex the tube for 15 sec.

16. Place the tubes on the MagListo™ Magnetic Separation Rack. Attach the magnet plate and invert the

rack gently 3 to 4 times until the beads bind tightly to the magnet.

17. Without removing the tube from the MagListo™ Magnetic Separation Rack, transfer supernatant

containing DNA carefully to a new sterile microcentrifuge tube.

18. Discard the tubes with the remaining Magnetic Nano Bead pellet. Do not reuse the beads.

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Summary of Reagents Volume in Each Step

Step Buffer Volume Page

Gel Solubilization Buffer ① (Gel Solubilization) ~ 1200 μl P. 8

Bead Binding Magnetic Nano Bead - DNA 100 μl P. 8

1st Washing Buffer ② (1

st Washing) 700 μl P. 9

2nd

Washing Buffer ③ (2nd

Washing) 700 μl P. 10

3rd Washing Buffer ④ (3

nd Washing) 700 μl P. 10

Elution Buffer ⑤ (Elution) 30 - 50 μl P. 10

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XIV. Appendix

Troubleshooting guide

This troubleshooting guide will help you to solve problem that may arise during DNA extraction. For

other technical assistance or more information, please contact our technical assistance team.

Comments and suggestions

Low recovery of DNA

fragment

Buffers or other reagents may have been exposed to external factors that may

have reduced its quality. Please make sure that reagents are stored at room

temperature at all times upon arrival and that all reagent bottles are closed

tightly, in order to preserve pH and stability, and to avoid contamination.

High pH conditions can reduce the overall yield due to incorrect binding

conditions. This can be examined by using Buffer ⓛ (Gel Solubilization), which

has a pH indicator. If the color is yellow but it turns to red or orange, then the pH

is out of range. In this case, several drops of sodium acetate buffer are

recommended to adjust the pH of the solution appropriately.

Incomplete dissolving DNA of the gel slices gives lower yield. DNA can be

remained in any undissolved agarose gel. Provide enough time for gel slice to be

dissolved completely.

Elution may have been incomplete. Please extend incubation time up to 3

minutes at elution step to improve the yield. In addition, make sure that Magnetic

Nano Beads are suspended completely in the eluting solution during incubation.

Some of Magnetic Nano Bead pellet may have been lost while discarding

solution. Check that all of the Nano Beads have bound tightly to the magnet

when you discard supernatant.

Insufficient shaking or vortexing during lysis step may lead to low DNA yield than

expected. Shake or mix with a vortex mixer sufficiently during incubation step.

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Excessively clustered

Magnetic Nano Bead

Excess amount of starting sample is used to extract DNA. Appropriate amount

of starting material (see “Kit Specification” in page 4) should be used for

efficient extraction of fragment DNA.

Presence of a white

precipitate in buffers

A white precipitate may form in Buffer ⓛ (Gel Solubilization) due to prolonged

storage at low temperatures. Incubate Buffer ⓛ at 60℃ to dissolve any

precipitate in the buffer.

Flotation of extracted

DNA when loaded on

an agarose gel

Floating of DNA on an agarose gel is caused by the remaining ethanol in the

eluted DNA. Ensure that the 3rd Washing (ethanol removing) step in the protocol

is properly performed. Remaining ethanol may also interrupt the enzymatic

reaction.

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Experimental data

Figure 1. Comparison of Fragment DNA from TBE agarose gel purified with MagListo™ 5M Gel Extraction Kit

and competitor’s kit (Single Column Type)

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

M: Bioneer 1 Kb ladder

1-2: 500 bp DNA purified with Bioneer MagListo™ 5M Gel Extraction Kit

3-4: 500 bp DNA purified with competitor Q kit

5: Control 500 bp DNA

6-7: 3 kb DNA purified with Bioneer MagListo™ 5M Gel Extraction Kit

8-9: 3 kb DNA purified with competitor Q kit

10: Control 3 kb DNA

11-12: 5 kb DNA purified with Bioneer MagListo™ 5M Gel Extraction Kit

13-14: 5 kb DNA purified with competitor Q kit

15: Control 5 kb DNA

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XVII. Ordering Information

Cat no. Product Description Size

K-3601 MagListoTM 5M Plamid Extraction Kit, 100 reactions (mini) 1 kit

K-3600 MagListoTM 5M Plamid Extraction Kit, 500 reactions (mini) 1 kit

K-3603 MagListoTM 5M Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit

K-3605 MagListoTM 5M Plant Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit

K-3607 MagListoTM 5M Gel Extraction Kit, 100 reactions (mini) 1 kit

K-3609 MagListoTM 5M PCR Purification Kit, 100 reactions (mini) 1 kit

K-3611 MagListoTM 5M Cell Total RNA Extraction Kit, 100 reactions (mini) 1 kit

K-3613 MagListoTM 5M Tissue Total RNA Extraction Kit, 100 reactions (mini) 1 kit

K-3615 MagListoTM 5M Forensic Sample DNA Extraction Kit, 100 reactions (mini) 1 kit

K-3617 MagListoTM 5M Viral DNA / RNA Extraction Kit, 100 reactions (mini) 1 kit

K-3619 MagListoTM 5M Circulating Cell Free DNA Extraction Kit, 50 reactions (mini) 1 kit

TM-1000 MagListoTM-8Ch Magnetic Separation Rack 1 ml tube x 8 holes

TM-1010 MagListoTM-2 Magnetic Separation Rack 2 ml tube x 8 holes

TM-1020 MagListoTM-15 Magnetic Separation Rack 15 ml tube x 6 holes

TM-1030 MagListoTM-50 Magnetic Separation Rack 50 ml tube x 3 holes

HT-15-NG 1.5 ml microcentrifuge tube 500 ea / pk

HT-20-NG 2 ml microcentrifuge tube 500 ea / pk

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XIX. Explanation Symbols

Catalog

Number

Contains sufficient for

(n) tests USE BY

Batch code

Caution, consult

accompanying

documents

Temperature

Limitation

Manufacturer

Caution, Potential

Biohazard

DO NOT

REUSE

Consult Instruction

For Use