Can't freeze my comp cells!
prigge at biology.lsa.umich.edu
Tue Mar 28 20:29:24 EST 2000
On Tue, 28 Mar 2000, John Dixon wrote:
> Hi all, I am having a bit of grief with my competent cells at the mo. I
> usually make them by inocc'ing at start of day, spin down at OD 4-6 (by
> eye) and wash in icy 0.1M CaCl2 spin down and resusp in same. Eff 10e7
> to 10e8 which is more than sufficient for my day to day stuff.
> However, it's a pain in the proverbials to make new ones each day as it
> should be so easy to freeze and store them. But every time I try to
> freeze them, the next day the efficiency is below my detection level of
> 10e6. So whats the secret?
Hmmm, the only thing I can think of is making sure the cells don't warm up
when aliquoting. (Are you aliquoting them?) I move everything into a
cold room and try not to handle the aliquot tubes more than necessary to
prevent my hands from warming the tubes. Supposedly, a dry ice/ethanol
bath is best for freezing them, but I worry too much about ethanol
splashing into tubes and killing the cells. (I generally use liquid N2.)
Michael J. Prigge phone: (734) 647-4153
Department of Biology fax: (734) 647-0884
University of Michigan
830 North University
Ann Arbor, MI 48109-1048
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