- grow up single colony in 1.5 ml LB/antibiotic ovn @37°C
- pour into tube, spin for 30 sec
- decant supernatant and resuspend pellet in 100 ml lysis solution
- add 200 ml alkaline SDS, vortex well
- incubate for 5 min on ice
- add 150 ml high salt solution, vortex well
- incubate for ca. 10 min on ice
- spin for 10 min
- remove 400 ml supernatant and transfer to new tube prefilled with 400 ml isopropanole
- vortex well and keep tube on ice for 10 min
- spin for 10 min
- wash pellet with 70% EtOH
- vacuum dry pellet for 5 min and take up in 100 ml 1x TE/20 mg/ml RNAse A
- usually 1-2 ml is enough for digest (high-copy plasmid), keep DNA frozen at -20°C
- phenolize important preps if to be kept for a longer period of time (more than 4-6 months)
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Solutions: |
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lysis solution:
(freshly prepared) 7.55 ml H2O
2 ml 50% glucose
0.2 ml 0.5 M EDTA
0.25 ml 1 M Tris-HCl pH 8
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10 ml total |
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alkaline SDS:
(stable for 1 week)
7.6 ml H2O
2 ml 5% SDS
0.4 ml 5 N NaOH
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10 ml total |
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hight salt solution: 3 M NaOAc pH 5.2
(same solution as used in precipitating DNA) |
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Remarks:
Even if kept at -20° C 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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Preparing Overnight