Electroporation advice needed

Jarkko Kortesmaa jkortes at paju.oulu.fi
Fri Apr 21 04:03:22 EST 1995


I recently tried transforming E.coli by electroporation. Efficiency was 
not very high, only 1,5x10^6.

I haven't done this before and neither has anyone in your institute. (The 
device has been used with eukaryotic cells only.) This is why I would 
like any suggestions on what may have gone wrong, or on what should I try 
next. 

This is what I did:

The cells were prepared according to manufacturers (Bio-Rad) protocol.
(Grown in low-salt LB and washed 4 times with water and suspended in 10% 
glycerol.) 

Electroporation was also performed as suggested in the 
manual. (12,25 kV field strengt, pulse lengt was about 4,5 ms. Cuvettes 
had 2 mm gap. 40 microL cells and 1 microL sample. 1 ng of pUC19 was used as 
control.) 

Immediately after electroporation 960 microL of LB was added 
and the cells were grown in 15 ml Falcon tubes at 37C for 1h with shaking 
and then plated on LB-AMP plates.

Thanks,
--
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Jarkko Kortesmaa		Dept. of Biochemistry and Biocenter Oulu
jkortes at paju.oulu.fi		University of Oulu
				Finland					
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