Can't freeze my comp cells!

John Dixon jpcd100 at mole.bio.cam.ac.uk
Tue Mar 28 11:15:22 EST 2000


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.

Cheers
JD

-- 
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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