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M13/Phagemid DNA Extraction Protocol


Efficiency of the standard protocols(1,2) for production of single-stranded DNA may be improved through the use of PLG.(3)
  1. Propagate M13 phage by one of the standard procedures:
    1. For producing M13 ssDNA, mix 2.5 ml plating bacteria (57 hours, 50 ml culture; stored at 4 C for no more than 3 days) with 300 l M13 phage stock (1 x 1011 pfu/ml) and incubate 5 minutes at room temperature to allow phage to interact with the bacterial phage receptors. The phage-bacteria culture is added to 250 ml LB broth and incubated for 57 hours at 37C with sufficient agitation for good aeration of the culture.
    2. For producing phagemid ssDNA, suspend a colony of phagemid transformed bacteria in a 15 ml culture tube containing 3 ml LB broth. Helper phage M13K07 is added to a final concentration of 2 x 107 pfu/ml and the culture is incubated for 1.5 hours at 37C with sufficient agitation. Recover resultant supernatant to a fresh tube or bottle and re-centrifuge as above.
  2. Recover resultant supernatant to a fresh centrifuge tube, add 0.25 volume of 20% PEG 8000/2.5 M NaCl to the supernatant, mix thoroughly by repeated inversion, and incubate 20 minutes at room temperature. Centrifuge at 15,500 x g, 4C, for 20 minutes and discard the resultant supernatant. Allow any traces of supernatant to drain from the tube.
  3. Resuspend pellet in 50 l TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) for every 1 ml of supernatant (step 3) and transfer the entire sample to a pre-spun (1500 x g fo r 12 minutes) PLG 15 ml Light tube.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. Let stand for 1530 minutes at room temperature, and centrifuge for 20 minutes at 12,000 x g or greater at 4C.
  9. Wash pellet twice with 0.5 ml 70% Ethanol and air-dry.
  10. 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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