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Electroporation strange problem

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#1 biolab12



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Posted 23 December 2009 - 05:35 AM

Hi all,

I am trying to transform electrocompetent DH5 alpha cells with a standard plasmid . I use 40 ul of the cells and about 50ng DNA. Electroporator is from eppendorf. Conditions : 1450 V using 0.1 cm cuvettes (time constant I get is 5.4 ms ). After electroporation, I let the cells recover in LB for about 1 hr at 37 deg.

I normally centrifuge the sample and remove about 800ul LB and resuspend the cells in remaining 200ul and plate it on appropriate LB plates.

I have never had problem with this technique and always had high transformation efficiency. But recently with the new batch of electro competent cells, I see a strange white jelly like thing after I centrifuge my cells .I also have no colonies after transformation. I am guessing the cells are dead and perhaps it is the cell debris that I see after centrifugation. Is it because of the pulse from my electroporator?

Anybody had a similar problem or any suggestion?

#2 phage434



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Posted 23 December 2009 - 05:52 AM

Possibly, or the cells could be dead prior to electroporation. You can test this by serial dilution of the cells and plating on non-selective medium. Your electroporation voltage sounds high. I would normally expect around 1.1 to 1.3 KV for a 1 mm cuvette. I usually do recovery in SOB not LB, but this is almost certainly not your problem. I'd suggest just making new electrocompetent cells.

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