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Basic Plasmid DNA Isolation Protocol

PLG can be used in small and large scale protocols based on alkaline lysis procedure.(1,2,3)

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 process or precipitate the samples as per your protocols and procedures.
References
  1. Emanuel, J.R. (1992) Phase Lock Gel plasmid micropreps: direct insert screening, probe synthesis and sequencing within one day. Nucleic Acids Res. 20:625.
  2. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7:1513-1522.
  3. Murphy, N.R. and Hellwig, R. J. (1996) Improved nucleic acid organic extraction through use of a unique gel barrier matrix. BioTechniques 21:934-939.

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