In article <3875C9F4.C31D0AAC at ua.es>, Rafael Maldonado
<rmaldonado at ua.es> wrote:
> John Dixon wrote:
> > I used to regularly make >10E8 competency cells using the Inoue method
> > with XL1s and XL10s, but now I need DH10Bs and they just don't seem to
> > like the Inoue method in mine and a colleagues hands (10E6 or less in
> > four attempts). The glycerols stocks are new and if made competent by
> > CaCl2 method they work OK (~10E6 to 10E7). Any clues?
>> Inoue protocol is optimized for DH5a. It is expected that different
> strains will behave different in a chemical transformation protocol.
> Electroporation may be your better choice for high competence, since it
> is less strain dependent.
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).
John Dixon Lab 44 (1223) 334131
Wellcome/CRC Institute Fax 44 (1223) 334089
United Kingdom CB2 1QR e-m: jpcd100 at mole.bio.cam.ac.uk