Electroporation Improvements

Anthony Tomlinson tomlinson at pplros.demon.co.uk
Thu Sep 22 10:02:51 EST 1994


In article <574815148wnr at genesys.demon.co.uk> Duncan Clark,
Duncan at genesys.demon.co.uk writes:
>> live cells per 50ul sample = 7.5X10^7 (30X lower than literature)
>> cell survival after electroporation = near 100%
>> fraction of surviving cells which receive plasmid = 67%
>
>What happens if you concentrate the cells more. You should be taking 1 
>litre down by at least 500 fold ie 2ml. At your 1.5OD you should have 
>roughly 10e8 cells/ml so concn to 2ml should give you 2.5x10e9 in 
>50ul. How come you only have 7.5x10e7? 

The first couple of times I made electrocompetent cells, the supernatant
was pretty cloudy during washes, which I attribute to excessive cell
lysis in the water.  Basically I washed more quickly/deftly and was ultra
careful with keeping everything cold, and it worked OK.  If you are
concentrating as you should (see above) then cell death may be the
problem.

Anthony



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