TECHNICAL BRIEF Automated Liquid Handling

Introduction

With the expansion of genomics applications, there is an increased need to automate routine manual protocols for increased throughput and sample volume without sacrificing data quality. The Zephyr® Molecular Biology Workstation offers a compact solution for automating molecular biology processes such as nucleic acid purification, DNA/RNA extraction, reaction setup and DNA/RNA normalization without compromising functionality or system flexibility. An intuitive graphics interface and straightforward protocols allow even occasional users to achieve highly accurate, reproducible results.

Here we describe the use of the Zephyr Molecular Biology Workstation in automating sequencing reaction cleanup with Agencourt® CleanSEQ® dye terminator removal kit (CAT# A29151). Using this protocol, sequencing reactions are purified with Solid Phase Reversible Immobilization (SPRI) paramagnetic

bead technology. The results demonstrate the efficient purification of sequencing reactions using SPRI Technology on the Zephyr Molecular Biology Workstation (Figures 1 and 2; Table 1).

Quality and Typical Chromatograms

25 μL single primer extension sequencing reactions were set up containing 10-50 fmol PCR product or 500 ng pGEM Plasmid template DNA, 5 μL ABI 5X sequencing Buffer, 12.5 pmol primer, 2.5 μL BigDyeTerminator Premix (v3.1) and nuclease free water. The 25 μL reactions were purified with the Agencourt® CleanSEQ® Sequencing Reaction Cleanup Kit on the Zephyr Molecular Biology Workstation. The purified sequencing products were eluted into 40 μL nuclease free water and directly injected into an ABI 7900 DNA Analyzer (www.genewiz.com).

Automation of the Agencourt® CleanSEQ® Sequencing Reaction Cleanup Kit

Figure 1. Improved signal and reduced background result in better base calling. Shown is a typical section of sequence electropherogram before and after reaction cleanup.

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Conclusion

Sequencing reactions can be purified on the Zephyr Molecular Biology Workstation using SPRI beads in a 96-well format using the Agencourt® CleanSEQ® dye terminator removal kit in approximately 30 minutes. Excess dyes and salts from the starting sequencing reaction are efficiently removed. High quality sequence data featuring long read lengths were consistently achieved. The process is simple, fast and fully automated.

Figure 2. Typical electropherogram is displayed yielding QV20+ Score of >1000 bp demonstrating sequencing performance of improved quality and reliable base calling.

Score QV20

PCR Product (~70 0bp) PRE 15 108

POST 46 732

PRE 19 236

POST 46 729

pG EM Plasmid PRE 17 239

POST 48 1031

PRE 19 239

POST 47 1014

Table 1. Improved quality scores and read lengths of sequencing reaction products.

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Specifications

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%

Included with your Zephyr Molecular Biology Workstation:

• PC controller and monitor• Vacuum filtration station and pump• Waste bottle• Plate/lid gripper• PING No Contact Clog Detection• Zephyr Molecular Biology Workstation GUI and Maestro software• Startup Kit

Volume Range

25.5 Inches (648 mm)

29.5 Inches

Deck Configuration

Part No. Description

125282 Zephyr Molecular Biology Workstation with HV Head - 110V

125283 Zephyr Molecular Biology Workstation with HV Head - 220V

119728 (Optional) Zephyr Environmental Enclosure

122000 LabChip® GX - Automated DNA/RNA CE Separation