High-throughput isolation of ultra-pure plasmid DNA by a robotic system

Abstract

Background: With the availability of complete genomes, a systematic inventory of cellular processes becomes achievable. This requires assessing the function of all individual genes. Transfection of plasmid DNA into cell culture cells is an essential technique for this aim as it allows functional overexpression or downregulation of genes. While many robotic systems isolate plasmids for sequencing purposes, for more demanding applications such as transfections there is a shortage of robots for the high-throughput isolation of plasmid DNA.

Results: Here we describe a custom-made, automated device, which uses a special protocol to isolate plasmid DNAs with a purity sufficient for efficient transfections into mammalian cells. Approximately 1,600 ultra pure plasmids can be isolated in a 96-well plate format within 12 hours. As a unique feature the robot comprises the integration of a centrifuge instead of expensive columns, the use of a custom-made pipetting head with a movable gripper, especially designed shaking platforms and an acetone wash facility.

Conclusion: Using this robot we demonstrate how centrifugation steps with multiple precipitations, most notably through a precipitation step of SDS in isopropanol, lead to high purity plasmid DNA and make possible high-throughput transfections into mammalian cells for functional gene annotations.

Figure 1

Layout of the DNA isolation robot (A) and photographic view of the machine (B). Ax: Rails for x-motion of the pipetting and gripper head; Ay: Rail for y-motion of the head; Az: Rail for z-motion of the head; A1, B1: Platforms with heater and shaker for 96 fold deep-well-plates (DWPs); A2, B2, A3, B3: Platforms with shaker for 96-fold DWPs; Ae, Be: Static platforms for 96-well microtiter-plates (MTP); A4, B4: thermo-isolated DWP-platforms cooled to 4°C; A-20, B-20: thermo-isolated DWP-platforms with lid cooled to -20°C; C: Centrifuge with 4-fold rotor; Ca: Loading position of the centrifuge; P1, P2: Reservoir of pipetting fluid (Room temp); P3: Reservoir of pipetting fluid (4°C); P4, P5: Reservoir of pipetting fluid with integrated stirrer (room temp); P6: Reservoir of pipetting fluid with heater up to 80°C; Pk: Movable 96-fold pipetting head; Gp: Gripper integrated into the movable head for transporting DWPs; S1: Platform for a 96-fold DWP for loading and withdrawing acetone; S1a: Tiltable 96-fold pipetting head for supplying acetone; S1b: Tiltable 96-fold pipetting head for withdrawing acetone; S2: Platform for a 96-fold DWP for withdrawing the bacteria medium; S2a: Tiltable 96-fold pipetting head for withdrawing fluids; V: Ventilation for removing acetone vapour; W: Parallel washer for the tips of the 96-fold pipetting head.

Figure 2

Plasmid yield of a robot run (A) and comparison of transfection efficiencies of plasmid DNAs isolated by the robot and a commercial kit (B). (A) pLantern plasmid DNA was extracted by the robotic system. Probes were chosen randomly from four different plates of processes A and B, 10 μl of plasmid solution per slot were applied to a 1% agarose gel and visualised by ethidium bromide staining. Marker lane: 4 μl Smart Ladder (Eurogentec). The highest band (10 kb) is 80 ng, the band at 3 kb corresponds to the lowest plasmid band and is 24 ng. (B) Transfection of column-isolated GFP plasmid DNA versus robot-isolated plasmid DNA. 293 cells were transfected with equal amounts of DNA in a 24-well format. After 30 hours phase contrast and fluorescent microphotographs were taken at a 100fold magnification with a Zeiss Axiovert S100TV equipped with a FITC-filter. Arrows denote damaged cells in the transfections.