Large-Scale Isolation of Plasmid DNA
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实验步骤
1. Inoculate 200 ml of L2 containing appropriate selective agent with a single colony or culture containing the desired plasmid. Grow at 37 degrees overnight.
2. Spin down cells at 5000 rpm for 15 minutes in Sepcor bottles.
3. Carefully pour off supernatant and aspirate off any media remaining.
4. Resuspend pellet in 4 ml of ice-cold GTE by pipetting up and down. Let stand 5 minutes at room temperature.
Be sure to completely resuspend cells. Avoid creating bubbles.
5. Transfer to 50 ml conical tube (Corning). Add 8 ml of freshly prepared NaOH/SDS solution and mix by inverting. Do not vortex. Let stand on ice for 5 minutes.
6. Add 6 ml of ice-cold KOAc solution and mix by inverting rapidly. Let stand on ice for 5 minutes.
7. Transfer to 2 Oak Ridge tubes and centrifuge at 11,000 rpm for 20 minutes at 4 degrees. The pellet formed in this step will contain most of the chromosomal DNA, SDS-protein complexes, and other cellular debris.
8. Transfer the supernatant in each tube into 2 Oak Ridge tubes, for a total of 4 tubes. Add an equal volume of phenol/chloroform to each tube and mix by vortexing. Centrifuge for 5 minutes at 10,000 rpm and transfer the upper, aqueous layer to a fresh Oak Ridge tube.
9. Add 2 volumes of ethanol, mix by vortexing, and let stand at room temperature for 5 minutes.
10. Centrifuge for 10 minutes at 10,000 rpm.
11. Pour off supernatant and wash with 1 ml of ice-cold 70% ethanol. Be careful, as the pellet often loosens from the tube upon washing.
12. Dry pellet by air and resuspend in TE w/RNase.
Note: The volume in which this pellet is resuspended in is dependent upon the intended use of the plasmid DNA. If the DNA is to be further purified by CsCl gradient centrifugation, then the DNA must be suspended in a final volume of 4 ml TE, no RNase being necessary.
