Efficiency of (electro)transformation in Salmonella
(by cc122 from mole.bio.cam.ac.uk)
Fri Nov 28 11:33:34 EST 2008
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
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