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Efficiency of (electro)transformation in Salmonella

Efficiency of (electro)transformation in Salmonella - Protocols and Methods Forum

Efficiency of (electro)transformation in Salmonella - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 11-28-2008, 04:33 PM
C Coward
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Default Efficiency of (electro)transformation in Salmonella



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@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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Old 11-28-2008, 05:11 PM
DK
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Default Efficiency of (electro)transformation in Salmonella

In article <ggp6gu$qo2$[Only registered users see links. ].cam.ac.uk>, C Coward <[Only registered users see links. ].cam.ac.uk> wrote:

1. Try higher cell concentration. Typical E.coli preps are 1 L culture -->
resuspend in 2 ml final, i.e. 500X. Yours is only 50X.
2. Vary voltage. E.g. 1.8, 2.2, 2.4 kV for 2 mm gap, 1.4,1.6,1.8 kV
for 1 mm gap.
3. Try 7% and 10% DMSO in place of glycerol.
4. Try growing cultures at 20C instead of 37. Gives a 10X boost
for E.coli.
5. Try including 50 mM DTT in washes (will require voltage
optimization since when the cell wall is weakened the electric
filed intensity needs to be reduced).

DK

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efficiency , electrotransformation , salmonella


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