Plant & Animal Genome IX Conference

Poster: Sequencing & EST
P73

AN AUTOMATED HIGH THROUGHPUT PLASMID PURIFICATION METHOD USING MAGNETIC PARTICLES THAT PROVIDES HIGH QUALITY TEMPLATE FOR ABI SEQUENCING METHOD

Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711

Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711-5399, USA We have developed a method for automated, high-throughput purification of sequencing-grade plasmid from 0.5 to 1.5 ml cultures of Esherichia coli containing a high copy plasmid, pGEM3Zf+. The method has been developed with a Beckman BioMek 2000, and adapted to other robotic systems, such as the Tecan Genesis150, Tomtec Quadra 96, and the BioMek FX. The method uses magnetic particles to clear the lysate and bind the plasmid. Starting with 1 to 8 OD₆₀₀ cell pellets in deep-well culture plates, the process requires no manual interventions and takes 90 minutes to complete. Yields obtained from 4.0 OD₆₀₀ of E. coli DH5 containing pGEM3Zf+ ranged from 6-8 ug of plasmid at a concentration sufficient to be used in a sequencing reaction without a concentration step. Using the ABI Prism Model 377 sequencer, DNA templates prepared by this method were sequenced using the ABI Big Dye^[tm] Terminator kits. The Big Dye^[tm] Terminator chemistry routinely yielded 700 base reads with >98% accuracy. Sequencing on an ABI Model 3700 sequencer routinely gave 600 base reads with >98% accuracy.