Does Inoue method work on DH10Bs?

ChenHA hachen at bun.com
Sun Jan 9 19:29:44 EST 2000


John Dixon wrote:

> Thanks, but I was hoping the two strains weren't that different. Any
> suggestions as to what parameters are worth varying first? (I have
> tried OD600 from 0.4 to 0.7 with little effect).
>
> I don't really want much higher than 10E7 competency and the
> hassle/expense/variability of electroporation, just the convenience of
> making a large batch to have in the -80, so if anyone could recommend a
> good freezing buffer for CaCl2 treated cells I would also be grateful
> (10% glycerol or 7% DMSO doesn't seem to help).
>

Snap freeze in liquid nitrogen.  You won't need your normal freezing buffer
but I always used them anyway (I use DMSO).  As for the transformation
efficiency, did you try varying heat-shock time?  You can also check on the
Inoue paper and see how they vary the different constituents in the buffer.
There are, of course, many other methods for making competent cells you could
try.

>
> 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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