Transformation of Helicobacter Pylori

Reference:

O’Toole et al, 1994, Mol. Microbiol. 14: 691-703.

Method:

1. Plate-grown H. pylori are resuspended in BHI to an OD₆₀₀ of 0.6.
2. 1 µg of DNA is added and incubated for 3 h at 37ºC (in a CO₂ incubator).
3. The cells are then spun down and resuspended in 100ul of BHI.
4. The entire 100 µl is spread onto a chocolate blood agar (CBA) plate (no antibiotic) and incubated for 24 h.
5. After 24 h the cells are scraped or washed from the plate and replated on CBA with antibiotic. Single colonies are restreaked for further analysis.
6. PCR can then be used to determine if the gene replacements have occurred as predicted and to confirm that no other rearrangements have occurred.