( ...)Basic Plasmid DNA Isolation Protocol

The basic protocol given here works with E. coli cultures grown for 1214 hours in up to 500 ml of amplified or non-amplified LB or DYT, or in 250 ml Terrific Broth.

1. Pellet bacteria from the culture at 10,000 x g for 5 minutes at room temperature.
   
2. Resuspend bacterial pellet in a total of 15 ml 25 mM Tris-Cl/10 mM EDTA (pH 8.0). Pipet up and down or vortex as necessary to fully resuspend the bacteria.
   
3. Add 15 ml room temperature 0.2 N NaOH/1.0% SDS to the suspension. Mix thoroughly by repeated gentle inversion. Do not vortex.
4. Add 15 ml ice-cold 7.5 M Ammonium Acetate to the lysate. Mix thoroughly by repeated gentle inversion. Do not vortex.
   
5. Centrifuge at 15,500 x g for 30 minutes at 4C.
   
6. Recover resulting supernatant and divide it between 2 pre-spun (1500 x g for 23 minutes) PLG 50 ml Heavy tubes.
7. Add 20 ml Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1) to the sample in each PLG 50 ml Heavy tube and mix thoroughly by repeated inversion. Do not vortex. Centrifuge at 1500 x g for 5 minutes to separate the phases.
8. Recover resultant aqueous phases to fresh pre-spun PLG 50 ml Heavy tubes and repeat step 7 above.
9. Recover resultant aqueous phases to fresh pre-spun PLG 50 ml Heavy tubes. Add 20 ml Chloroform-Isoamyl Alcohol (CI, 24:1) and extract as in step 7 above.
   
10. Recover resultant aqueous phases to suitable tubes and
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