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Preparing competent E. coli

Danuta Bois danutabois at delphi.com
Sun Jul 10 10:32:58 EST 1994


Dear Netters,
I've been trying to make E. coli competent for transformation
using a modified chemical method of Hanahan (prococol I in Molecular
Cloning. A Laboratory Manual, by Sambrook et al.). They say you should get
efficiencies of transformation greater than 5x10E8 for DH1, DH5 and MM294.
I've tried it with DH5alpha and HB101, and the best that I've gotten was
2x10E6. DH5alpha may have been an old culture (I got it from another lab),
but HB101 was taken from a tube of frozen high efficiency cells from Promega
and grown overnight in SOB medium, then diluted 100x with SOB + 20mM MgSO4
and grown up to <0.4 OD600. Would anyone have an idea what my problem is and
how to fix it? I would appreciate any help.





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