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1.grow up single colony in 1.5 ml LB/antibiotic ovn @37℃ . 2.pour into tube, spin for 30 sec . 3.decant supernatant and resuspend pellet in 100 ml lysis solution . 4.add 200 ml alkaline SDS, vortex well . 5.incubate for 5 min on ice . 6.add 150 ml high salt solution, vortex well. 7.incubate for ca. 10 min on ice . 8.spin for 10 min. 9.remove 400 ml supernatant and transfer to new tube prefilled with 400 ml isopropanole. 10.vortex well and keep tube on ice for 10 min . 11.spin for 10 min . 12.wash pellet with 70% EtOH . 13.vacuum dry pellet for 5 min and take up in 100 ml 1x TE/20 mg/ml RNAse A . 14.usually 1-2 ml is enough for digest (high-copy plasmid), keep DNA frozen at -20℃. 15.phenolize important preps if to be kept for a longer period of time (more than 4-6 months).
hight salt solution: 3 M NaOAc pH 5.2 (same solution as used in precipitating DNA) Remarks: Even if kept at -20℃ DNA from this quality will degrade over a period of a year or so when not phenolized carefully (ca. 3 x)! Take care that no interphase remains. |
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