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


CONCLUSIONS
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.
Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany. Kachels@web.de
Volker
V
Kachel
Georg
G
Sindelar
Stefan
S
Grimm
D005591:Chemical Fractionation / Q000295:instrumentation* / Q000379:methods; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D010957:Plasmids / Q000302:isolation & purification*; D012371:Robotics / Q000295:instrumentation* / Q000379:methods
D004269:DNA, Bacterial