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Methods:P1 transduction

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Contents

Purpose

Lysates of P1 includes particles that are packed with the bacterial chromosome. It has no specific attachment site on the bacterial chromosome so the fragment will recombine into the host chromosome. [1]

This method is used to move alleles from 1 strain to another using P1.

For a more descriptive explanation of this method, check out the Transduction EcoliWiki page.

Materials Needed

  • Donor and Recipient strains
  • P1vir lysate
  • Sterile 18x150 mm test tubes
  • Sterile 13x100 mm test tubes
  • Growth medium
  • 50mM CaCl2
  • Selective growth medium containing 20 mM Na Citrate

Protocol

  • Day 1

Streak out donor and recipient strains from freezer stocks

  • Day 2

Prepare a lysate by growing P1vir on the donor strain
Start overnight of recipient strain

  • Day 3

Perform transduction and plate on selective medium

  • Day 4

Colony purify transductants on selective medium containing 20 mM Na Citrate

  • Day 5

Screen colonies

Making the P1 Lysate

  • Inoculate LB with colony of donor strain
  • Grow at 37C to ~1x108 cells/ml (barely turbid)
  • Add 1/9 volume of 50 mM CaCl2
  • Add 20μl P1 vir
  • Grow at 37C with good aeration until lysis occurs
  • Add 5 drops CHCl3
  • Shake for 5 minutes
  • Spin out debris for 10 minutes in a tabletop centrifuge
  • Repeat centrifugation
  • Store supernatant at 4C in a screw cap tube with 2 drops CHCl3 in the dark

Doing the P1 Transduction

  • Grow overnight of recipient strain
  • Add 1/9 volume of 50mM CaCl2 in a sterile 13x100mm test tube
  • Samples are as follows:
    • 1. 0.2ml recipient cells - phage
    • 2. 0.2ml recipient cells + 0.05ml P1/vir grown on donor strain
  • Incubate standing for 20 minutes in a 37C water bath
  • Add 0.1 ml 1M Na-citrate and 0.7ml LB
  • Incubate standing for 40 more minutes
  • Plate 0.2ml on selective plates containing 20mM Na-Citrate
    • 3. also plate phage only control- spot 20μL of P1vir lysate

Notes

For ts strains, incubations may be done at 30C

If plates were made without Na- citrate, you can spread 0.1ml 1M Na-citrate on the plate

recA strains

Getting your marker into the recipient strain requires homologous recombination, which means that recombination deficient strains don't work. Lucigen sells a plasmid, pInvRecA carrying recA on a ts replicon (see manual (pdf))

References

See Help:References for how to manage references in EcoliWiki.

  1. Miller JH. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press,U.S. (31 Dec 1972)