Can't freeze my comp cells!
tiredkhan at yahoo.com
Mon Apr 10 23:53:34 EST 2000
I suggest that you keep with the Inoue method. The efficiency should be much
higher after freezing, as the freezing step is part of the process of making
high efficiency competent cells by this method. It would be good to know why
some labs find this reliable and consistent whereas others find that they
can't freeze without losing efficiency. It's probably down to just one small
thing, but what is it? Perhaps you could try using the highest quality water
that is available, and use brand new, rinsed plasticware throughout the
whole procedure so that your cells never see any traces of detergent.
"John Dixon" <jpcd100 at mole.bio.cam.ac.uk> wrote in message
news:280320001715222755%jpcd100 at mole.bio.cam.ac.uk...
> 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.
> 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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