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To satisfy demands for increasing numbers of purified plasmids, several different quasi-automated approaches have been developed. Our objective was to completely automate a commercially available cartridge procedure to isolate plasmids starting from 96 cultures in deepwell microplates. A script (Gemini 3.1) written for a Genesis RSP 8/150 with ROMA (TECAN AG) pipetting station directed the fully automated plasmid isolation procedure (QIAwell 96 Ultra plasmid isolation kit, QIAGEN). Liquids were handled using either disposable tips or washable probes while movement of all components was done by the ROMA. The Genesis was equipped with computer- regulated vacuum source, plate/cartridge storage hotel, 100 and 200 mL reagent reservoirs, and disposable pipet tip tray.
We evaluated the performance of our procedure by using two different plasmids (pUC18 and pPCR:CAM) transfected into two host strains of E. coli. Purified plasmids extracted from 96 wells of pUC18 culture gave an average A260/A280 of 1.86 +/- 0.08. Electrophoresed aliquots of each plasmid sample showed > 95% of each sample were supercoiled with no genomic DNA from E. coli. detected in any of the samples. To detect for possible cross contamination, we extracted a microplate with two different transfected cultures (pUC18 and pPCR:CAM) placed in alternating wells of a 96 position deepwell microplate. No occurrence of neighboring (contaminating) plasmid were observed for 24 samples when electrophoresed in agarose. Identical results were obtained when a PCR-based approach was used to detect for cross-contamination.
Conclusion: We developed a fully automated procedure that yields high purity plasmids that are free of host genomic DNA, are PCR-ready, and are not cross contaminated by neighboring plasmids.