Freezing competent bugs...

Rafa Maldonado Rafael at genetics.med.utah.edu
Thu May 12 18:32:19 EST 1994


Hi David!

You are right: tyhe better system for freezing bacteria is 7% DMSO. The
viability of frozen cells with DMSO is better than with glycerol, and
DMSO isn't so messy.
I usually add 70ul in 1 ml of LB culture to keep my collection. I have
never found a significant level of death in my collection (even 7 years
of deep freezing at -70, and including any possible error in my
pipetmans!), so your problem should be a different one...
I don't understand what your are doing. First, using the Inoue
protocol, you don't have to resuspend the cells in LB to keep them
frozen. If you do so, you'll lose all the competence! Add the DMSO
directly to the cell suspension in TB, and keep at -70. When the cells
are resuspended in LB is to keep in collection. Maybe you are not
explaining very well the procedure.
Second, one hour at 37 is enough for detecting growth, but not in this
conditions. Normally, I don't see increasing in the OD doing my
transformations.
Finally, don't forget to use liquid nitrogen to freeze the competent
cells. But, again, to keep in collection, I usually put the vials
directly in the freezer. 
I think the concentration of DMSO can be very tolerant. I mean, with 8
or 6% the cells are not going to die. But, if you pipets have such
error (circa 15%), recalibrate them!
Please, post if you have more questions...

Rafa

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



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