Efficiency of (electro)transformation in Salmonella

C Coward via methods%40net.bio.net (by cc122 from mole.bio.cam.ac.uk)
Fri Nov 28 11:33:34 EST 2008


Hello all,

I'm after opinions/advice on how to get the best transformation efficiency 
in Salmonella (enteritidis).

I'm currently using electroporation but am routinely getting efficiencies 
around 10^4 cf/ug of DNA. I don't think restriction systems would be 
reducing my efficiency as the plasmid DNA is prepared from a Salmonella 
strain (I haven't tested this though, the plasmid DNA is coming from S. 
typhimurium LB5010). I do need to test the efficiency of re-introducing my 
plasmid preps into LB5010.

I could really do with higher efficiency so any tips?

My protcol is as follows

1. Grow a starter O/N in LB from 37C
2. Inoculate 20ml with 100ul of the starter, grow @37C to OD~0.5
3. Wash 1X in 20ml ice-cold dH2O
4. Pellet and wash 1X in 1ml ice-cold dH2O
5. Pellet and wash 2X in 1ml ice-cold 10% glycerol
6. Resuspend in 400ul 10% glycerol, aliquot in 100ul lots, store at -80
7. Electroporate using 2mm gap cuvettes, 2kV, 25uF, 200ohm (pulse time 
around 5ms)

Cheers!



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