Can't freeze my comp cells!
hzhen at freeuk.com
Tue Mar 28 19:31:46 EST 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?
> I have tried the following:-
> 1) No additives - just 0.1M CaCl2
> 2) 10% glycerol
> 3) 7% DMSO
> 4) freezing by transfer to -80
> 5) freezing by immersing in liquid N2
> 6) using Inoue type buffer instead of 0.1M CaCl2
> 7) using Z-comp buffer (Zymogenetics)
> But no joy - efficiency dropped to less than 10e6 every time.
> Any clues would be appreciated. Even of the "we use DMSO - works fine"
> variety, just so I know which options to concentrate on.
Doesn't sound like you have done anything wrong. I normally
use Inoue method, add 7% DMSO, snap freeze in liquid N2, and
store in liquid N2. To defrost, I keep the frozen cells on
ice for 30 minutes before adding to DNA. The cells are good
for at least 6 months (don't know about longer period
because the cells get used up rather quickly), and they
always come out perfect. I think if you read the literature,
freezing is supposed to make the competent cells more
competent. Unless you are using some funny strains or
haven't been too gentle with them...
Incidentally, keeping the cells overnight on ice is supposed
to increase the transformation efficiency (by the CaCl2
method), so if you continue to have problems, you can use
your freshly prepared cells for at least two days without
> John Dixon Lab 44 (1223) 334131
> Wellcome/CRC Institute Fax 44 (1223) 334089
> Cambridge University
> United Kingdom CB2 1QR e-m: jpcd100 at mole.bio.cam.ac.uk
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