( James Batchelor Lynn Jordan...)High Throughput Plasmid DNA Preparation Using A Magnetic Bead Kit On An Automated Workstation

James Batchelor, Lynn Jordan, and Ed Alderman
Applied Science and Technology Department, Caliper Life Sciences, Inc., Hopkinton, MA 01748

Karen Heins and Olaf Stelling
Agencourt Bioscience Corporation, 100 Cummings Center, Suite 107J, Beverly, MA 01915

Introduction

The goal for plasmid purification is isolation of nucleic acid from cellular material. This can be accomplished through the use of the SPRI-based paramagnetic microparticles and an external ring magnet plate. These microparticles are encapsulated with a ligand that binds nucleic acids. Once bound, the microparticles are drawn toward the wall of the microplate by a separate, external ring magnet plate. Waste material can be withdrawn from the well leaving the particles (and nucleic acid) behind. The particles are washed and the nucleic acid eluted from the microparticles.

This technology lends itself to high throughput environments. Centrifuges and vacuum pumps with filters are not needed to separate the nucleic acid from the waste.

Materials and Methods

Instrumentation:

Liquid transfers were performed with a Sciclone ALH 3000 fitted with a High Volume Head and using 100 μL and 200 μL Automation Certified Pipette Tips. A deck mounted Microplate Shaker (CaliperLS), was used for bead resuspension.

Microplates:

- 96 well 1.1 mL Culture Block (Marsh #DW9611)
- 96 well round bottom microplate (Corning #3765)
- 96 well Ring Magnet Plate (Agencourt P/N 000219)

Reagents:

- CosMCPrep Kit (Agencourt P/N 001046)
- Contains Resuspension/Elution, Lysis, Neutralization Buffer and Microparticles in binding buffer
- Isopropanol - 70% EtOH

Starting Materials:

- DH10B Cells
- 1 Rack of 100 μL tips per sample plate
- 1 Rack of 200 μL tips per sample plate

Sciclone Procedure

Methodology

1. Attach 100 μL tips
2 . Add 95 μL Resuspension Solution and shake @ 1200-1500 rpm for 4 minutes
3. Add 100 μL Lysis Solution and shake @ 800-1000 rpm for 5 minutes
4. Add 100 μL Neutralization Solution and shake @ 800-1000 rpm for 10 minutes
5. Slowly dispense 150 μL of air to gently bubble material away from the tips. This is accomplished by lowering the tips to a height just below the top of the well, then moving back to the 12 o'clock position and lowering the tips to the bottom of the well and dispensing the air
6. After a 3 second pause, aspirate 110 μL of Lysate and transfer to a 96-well round bottom microplate
7. Replace 100 μL tips with 200 μL tips
8. Transfer 80 μL Isopropyl Alcohol, 10 μL air gap, and 10 μL Microparticle Solution to the Lysate plate
9. Mix the solution using 10 aspirate/dispense cycles
10.Place the round bottom plate on the magnet plate and allow the microparticles to settle for 10 minutes
11. Transfer all of the solution to waste taking care to not disturb the ring of microparticles
12.Add 170 μL 70% Ethanol allowing to rest for 30 seconds and then transfer to waste
13.Repeat step 12 two more times

Results

The plasmids prepared on the Sciclone were compared with plasmids prepared on Agencourt's DNATrack System. Table 1 shows the sequencing data for each system. The devices compare very closely to each other, indicating the Sciclone procedure is performing closely to Agencourt's internal procedures. Figure 1 is a sequence trace from a sample purified on the Sciclone. Read lengths are comparable at 789 versus 779 bp for the DNATrack and the Sciclone, respectively. Each system gave a 94% pass rate out of 384 samplings (4 x 96 well plates). Nucleic acid yield for the two systems are roughly 16 ng/μL by PicoGreen binding analysis. Results from electrophoretic separation of the purified nucleic acid are presented in Figure 2.

Discussion

The Agencourt CosMCPrep Kit, Agencourt ring magnet plate, and Sciclone ALH 3000 can easily purify plasmids. The system does not require external hardware such as a centrifuge or vacuum pump/filter. The ring magnet locator easily allows complete removal of supernatant. Selective removal of lysate was accomplished by blowing air into the wells of the culture plate at the 12 o' clock position. Bubbles push residual cell material to the top of the well while leaving clean lysate at the bottom for easy transfer.

PicoGreen and sequencing data demonstrates that the CaliperLS Sciclone ALH performs comparably to Agencourt's procedures and hardware. Sequencing lengths and total yield were comparable on the two systems.