Plasmid Quickpreps

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.