Hancock Lab

Transposon Mutagenesis of Pseudomonas Aeruginosa

Categorized: Bacterial GeneticsFunctional Genomics

Method from Poole and Hancock. 1986. Molec Gen Genet. 202: 403-409.

Materials:

  • Strain with R68 ts::Tn501 (H526)
  • LB (high salt):1% tryptone, 0.5% yeast extract, 0.5% NaCl
  • 15 µg/ml HgCl2
  • Carbenicillin 500 µg/ml / tetracycline 100 µg/ml.

Method:

  1. Grow strain in LB broth + antibiotics (Carbenicillin and Tetracycline) overnight at 30ºC to select for R68ts::Tn501 plasmid.
  2. Do serial dilutions of 10-1, -2, -3, -4 of the overnight culture.
  3. Plate 0.05 ml or 0.1 ml of 10-3 and -4 dilutions onto LB plates (2% agar needed for replica plating) that contain 15 µg/ml HgCl2. Incubate at 42ºC overnight to select against R68ts::Tn501 plasmid. The HgCl2 selects for the jumping of Tn501 to the chromosome.
  4. Using sterile toothpicks, pick mutants onto fresh LB + HgCl2 plates. Incubate at 42ºC. overnight.
  5. Replicate plates with sterile velvet. Test mutants by colony blot lysis.

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