Freezing competent E coli

Christopher Coward Path cc122 at mbfs.bio.cam.ac.uk
Tue Mar 8 15:30:09 EST 1994


We routinely store electrocompetent cells at -70C in a glycerol solution (10%) after snap-freezing them in liquid nitrogen. Thawing before use is done
either on ice or at room temp. and I have not experienced any problems either
way, although I have not needed very high transformation efficiencies (yet!),
I have not been told of a problem with fast-thawing by other members of the group. I can't remember where hthe electrotransformation protocol was from, but
I think it was BioRad - preparation of competent cells results in the cells
from 1l of OD(600)=0.5-1.0 being resuspended in 2-3ml of 10% glycerol, this
is then aliquoted in 50microL amounts into microfuge tubes and snap-frozen.
We also routinely store non-competent cells in 10% glycerol (850microL
culture + 150microL sterile glycerol), mix well and freeze at -70 (no
snap freezing) - these cells are fine for extended periods of time, certainly
over 6 months.

Hope that's some help.

Chris



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