Method Note 014

PRODUCT CERTIFICATION

Automation of the Agencourt Ampure XP SPRI Bead PCR Purification Protocol on the Zephyr Genomics WorkstationIntroductionHere, we describe the use of the Zephyr Genomics Workstation in automating PCR purification with the Agencourt Ampure XP re-agent (Cat # A63880). With this system, PCR products are purified using magnetic bead-based Solid Phase Reversible Immobilization (SPRI) technology. The results confirm the expected yield and quality of PCR products purified using Agencourt Ampure XP technol-ogy on the Zephyr Genomics Workstation.

DNA Yield and Quality25 μL PCR reactions were purified with the Agencourt Ampure XP beads on the Zephyr Genomics Workstation. The purified PCR products were eluted in 40 μL molecular biology grade water. Average yields of amplified products of 79±12% and 86±15% were obtained for 640bp and 230bp amplicons respectively. Primers were consistently removed in the process and the purified PCR prod-ucts were used to generate high quality sequence data.

Figure 1a. Virtual Gel Image of unpurified PCR products. Samples were separated and automatically quantified using microfluidic capillary electro-phoresis on LabChip GX with the DNA 5K assay.

Figure 1b. Virtual Gel Image of purified PCR products. Products were eluted in molecular biology grade water and separated and automatically quantified using microfluidic capillary electrophoresis on LabChip GX with the DNA 5K assay.

014 - PRODUCT CERTIFICATION

Figure 2. LabChip GX DNA 5K Overlay Electropherogram Detail. Electropherogram trace before and after cleanup illustrates unincorperated dimer removal.

Figure 3. Sequencing quality report for PCR products before and after cleanup. Selected products were sent for sequencing (www.genewiz.com) in molecular biology grade water. Quality scores and read lengths were improved after cleanup.

ConclusionPCR products can be purified in a 96-well format using the Agencourt Ampure XP reagent (Cat #A63880). With this system, PCR products are purified using magnetic bead-based Solid Phase Reversible Immobilization (SPRI) technology in approximately 20 minutes. Average yields of amplified products of 79%±12% and 86%±15% were obtained for 640bp and 230bp amplicons respectively. Excess primers and salts from the starting PCR reaction mix were efficiently removed. The DNA is suitable for downstream applications, including primer extension sequencing. The process is simple, fast and fully automated on the Zephyr Genomics Workstation.

PRODUCT CERTIFICATION - 014

Figure 4. Sequencing Electropherogram Detail. Improvement in signal strength, background and base calling confidence can be seen in a representitive section of sequence after cleanup.

Post-Clean Up

Pre-Clean Up

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of Caliper Life Sciences, Inc. All other names are trademarks of their respective companies.

ZH-MN-014 Oct 10

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Performance

Volume range: High Volume Head (HVH) 1 - 200 µL

Precision: High Volume Head (HVH) 1 - 5 µL: CV <5% 5 - 200 µL: CV <2% 2 - 25 µL or 50 µL: CV 5%

Deck Configuration

Caliper Life SciencesPart No. Description

125282 Zephyr Genomics Workstation with HV Head - 110V

125283 Zephyr Genomics Workstation with HV Head - 220V

119728 Optional Zephyr Environmental Enclosure

122000 LabChip GX Analyzer

760541 LabChip XT Instrument

Caliper’s Other Genomic Solutions

LabChip GX LabChip XT

Additional applications which can be automated on the Zephyr Genomics Workstation:• LabChip GX sample preparation for RNA quality assessment or DNA fragment analysis

• LabChip XT for fast, automated nucleic acid fractionation accurately and reproducibly

Zephyr’s small foot print fits on to a standard lab bench, requiring no special table, while offering flexibility for molecular biology applications: • DNA extraction • RNA extraction • Plasmid isolation • PCR purification • Sequencing reaction clean-up • PCR setup • Sequencing reaction setup • DNA and RNA normalization • Partial or whole plate

What’s included with your Zephyr Genomics Workstation: • PC controller and monitor • Vacuum filtration station and pump • Waste bottle • Plate/lid gripper • “PING!” Ultrasonic detector • Genomics Workstation GUI and Maestro software • Startup Kit