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M13/Phagemid DNA Extraction Protocol

r 12 minutes) PLG 15 ml Light tube.
  • Add an equal volume of Tris-buffer saturated Phenol (pH 8.0) to the sample in the PLG 15 ml tube, and mix thoroughly by repeated inversion. Centrifuge at 1500 x g for 5 minutes to separate the phases.
  • Add an equal volume of Phenol-Chloroforrn-Isoamyl Alcohol (PCI, 25:24:1) to the sample in the same PLG 15 ml tube and extract and centrifuge as in step 5.
  • Add an equal volume of Chloroform-Isoamyl Alcohol (CI, 24:1) to the sample in the same PLG 15 ml tube and extract and centrifuge as in step 4.
  • Quantitatively transfer final aqueous phase to a 2 mL microcentrifuge tube, add 3 volumes of a 25:1 mixture of absolute Ethanol:3 M Sodium Acetate (pH 5.2), and mix well.
  • Let stand for 1530 minutes at room temperature, and centrifuge for 20 minutes at 12,000 x g or greater at 4C.
  • Wash pellet twice with 0.5 ml 70% Ethanol and air-dry.
  • Resuspend pellet in 2050 l TE, pH 8.0.
  • References
    1. Current Protocols in Molecular Biology, Volume I. (1989) Ausubel, F.M. et al. (eds.). John Wiley and Sons, New York, NY pp.1.14.1 - 1.15.4.
    2. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp.4.29-4.32.
    3. PLG Notes. (1992) Prime Report 4(2):

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