M13 Phage Growth and Single-Stranded DNA Preparation

  • Fiona M. Tomley
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 58)

Abstract

M13 bacteriophage has a single-stranded DNA (ssDNA) genome, and has proven an extremely useful vector from which to derive single-stranded templates for sequencing and site-directed mutagenesis. During infection of its host cell, the phage DNA replicates as a double-stranded intermediate from which the ssDNA containing phage particles are produced. Infected cells do not lyse, but instead phage particles are continuously released. Cells infected with M13 phage, however, have a longer replication cycle, which means that as the infection proceeds, the areas of slower-growing cells can be visualized as turbid plaques on the lawn of unaffected E. coli (1). Recombinant M13 phage can be cloned from well-separated plaques and used as a source of SSDNA.

References

  1. 1.
    Marvin, D. A. and Hohn, B. (1969) Filamentous bacterial viruses. Bacteriol. Rev. 33, 172–209.PubMedGoogle Scholar
  2. 2.
    Bankler, A. and Barrell, B. G. (1983) Shotgun DNA sequencing, in Techniques in the Life Sciences (Biochemistry), vol 85: Techniques in Nucleic Acid Biochemistry (Flavell, R. A., ed.), Elsevier, Amsterdam, pp. 1–34.Google Scholar
  3. 3.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Small-scale preparation of single-stranded bacteriophage M13 DNA, in Molecular Closing, A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 4.29–4.30.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Fiona M. Tomley
    • 1
  1. 1.Institute of Animal HealthComptonUK

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