Freezing competent E coli

Rafa Maldonado Rafael at genetics.med.utah.edu
Mon Mar 7 19:32:29 EST 1994


Hi Rick:
The best and easiest way to keep any E. coli in any state (competent or
for prolongued storage) is DMSO 7%. I think the protocol is in
Maniatis, but basically consist in putting the cells in ice (in your
favorite buffer or medium) and adding slowly the DMSO with gentle
shaking, incubate at 0C a couple of minutes and then deep the tubes in
N2. After that, you can put the cells in the deep freezer or store the
cells in N2. Glycerol inhibits chemical transformation (see discusion
about CaCl2 methods some weeks ago), even purest grade. DMSO desn't go
bad with the time (at least, in properties afecting the freezing
capabilities) and it's not necesary autoclave it.
I suppose that stored in N2 the cells live longer, but I think the
differences are no very big. I mean, I don't store any bacteria in N2.
However, you cannot use DMSO for electrocompetent cells, and I have
read (I don't remember where, maybe be in protocols from BTX, the make
of my electroporator) that electrocompetent cells are better frozen in
dry ice, I don't know why.
Freezing things directly in the freezer is no very good, but it depends
of what you want. I keep my stock of strains in this way: one vial
contains so many bacteria that is no very important losing a 10-20%.
But this difference is very important in competent cells, and the
competence is strongly afected if you don't freeze them in N2.
Dry ice is a good alternative if you don't have N2, but it's better use
a ethanol-dry ice beaker to faster freezing: mix etahnol with some dry
ice in a beaker or dewar vase and freeze the cells in a floater (as you
mention).
For thawing, I usually put the vials in ice, so they are thawed very
slowly. I have no experience in faster thawing.
That was all my experience. More suggestions will be apreciated.

Rafa

----------------------------------
Rafael Maldonado
Howard Hughes Medical Insitute
University of Utah
Rafael at genetics.med.utah.edu



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