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Methods:M13-mediated phagemid transduction

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Contents

Purpose

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Transfer of plasmids without transformation. Plasmids must contain a compatible origin of replication

Materials Needed

  • M13 rv-1 helper phage
  • phagemid-containing cells (e.g. from pZ150 or pZ152[1])
  • 2X YT media

Protocol

  1. prepare phage stock as in Methods:CTAB_M13_phagemid_prep
  2. pasteurize the phage stock by heating at 55-60°C for 15-20 minutes
  3. use 5 μl phage with 50 μl of a fresh overnight of a susceptible host
  4. incubate at 37 degrees C for 30 minutes to allow expression of antibiotic resistance genes
  5. plate on agar with appropriate antibiotics

Usage Examples

User notes

This method gives high enough transduction so that you can spot a few microliters of the transduction mix on the plate and streak for single colonies from the spot.

M13 transduction is somewhat mutagenic. Mutations in lambda cI plasmids occurred at approx 10-2 - 10-3 per transductant.

References

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  1. Zagursky, RJ & Berman, ML (1984) Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing. Gene 27 183-91 PubMed EcoliWiki page