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.