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).
| Solutions: | ||
|
lysis solution: (freshly prepared) 7.55 ml H2 O 2 ml 50% glucose 0.2 ml 0.5 M EDTA 0.25 ml 1 M Tris-HCl pH 8 ------ 10 ml total |
alkaline SDS: (stable for 1 week) 7.6 ml H2 O 2 ml 5% SDS 0.4 ml 5 N NaOH ------ 10 ml total |
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.
