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Miniprep for sequencing - (Dec/01/2006 )

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Try doing a Qiagen miniprep without using the columns, from a 5mL culture.

Pellet Bac in 1.5mL eppendorf, resusp in 300uL P1 (with RNAse), lyse in 300uL P2 (no need to inc @ RT for 5'), neutralize in 300uL P3, inc on ice for 10'. Centrifuge @ max speed for 8', Xfer SN to clean tube and recentrifuge. Xfer SN again to a fresh tube containing 630uL isopropanol. Vortex, spin @ max for 8. Wash w/70% EtOH, air dry for 5-10'. Reuspend in sterile/superclean milliQ water, ~20-30uL.

Works like a charm. I get at least 2 ug of DNA per prep with a 5kb plasmid, usually more. And my sequences are beautiful and often go out to 850+bp......

Good luck.


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