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[Boo]

Hi all,

I need some suggestions desperately on improving the electroporation efficiency of P. aeruginosa.

I have posted previously on problems with transposon mutagensis using the Epicentre Kan-2 kit. I think the problem I am having is a low efficiency of electroporation.

I only get 2 or 3 mutants, but these are confirmed to contain the kanamycin cassette by my PCR detection method.
I have tried lots of avenues...

Basically my method is:

Inoculate 90ml LB with a 10ml overnight culture.
Incubate for ~5 hours.
Wash cells three times in ice-cold hi pure water and resuspend in 1ml of 10% glycerol/ hi pure. This is using a bench-top centrifuge that isn't refrigerated - would this prevent efficient electroporation later on?

I always have a control electrocuvette (bugs only) to make sure the cuvette doesn't arc (ie cells are washed properly). I get around 10 to the 10 bugs to electroporate.
I have a brand-new kit therefore the transposon shouldn't be the problem.
I am confident that my kanamycin plates are working (500ug/ml).

Electroporation conditions are: Add 1ul transposon to 40ul cells. Electroporate at 25ìF, 200Ù and 250kV/cm for approx 5 s (using 0.2cm cuvettes, new each time).

The kits are expensive and I am already onto my second one (and now into the 2nd yr of my PhD!!!).

If anyone has any suggestions I would love to hear them!!

Also can anyone suggest an "electroporation control" I can try to test whether preparing my cells using my method makes them competent?


Thanks again.

Boo