Freezing competent E coli

Bernard Heymann bheymann at bragg.bio.purdue.edu
Sat Mar 12 22:30:19 EST 1994


In article <2lg8e9$itc at news.bu.edu>, rocket1 at bu.edu (Richard Near) wrote:

> I've been having difficulty in the storage of competent
> bugs for transformation either with CaCl2 or by 
> electroporation.  Questions are: Is it better to freeze
> in dry-ice-ethanol or snap freeze with liquid N2 (or
> can one gradually freeze things in a "Revco"?

I have been using 20 % glycerol for competent and non-competent cells now
for a long time. The one parameter that seems to affect both competency and
viability is the amount of time allowed for the glycerol to penetrate the
cells (presumable to be effective in preventing ice crystal formation). I
usually leave non-competent cells with 20 % glycerol for about an hour at
room temp, and competent cells on ice, before simply putting them in the
-70¡C freezer. I'll try DMSO next just to see how that compares.

-- 
Bernard Heymann
bheymann at bragg.bio.purdue.edu



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