Preparing competent E. coli
tan at aeolus.vmsmail.ethz.ch
Mon Jul 11 06:02:07 EST 1994
In article <nhl-100794141159 at 220.127.116.11> nhl at binkley.cs.mcgill.ca (Nancy
(referring to) article <9407101132591.DLITE.danutabois at delphi.com>
danutabois at delphi.com (Danuta Bois)
>>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
>I have a very hard time believing such high competencies. I know 10^5 and
>10^6 are possible, but the only time I've seen efficiencies higher than
>10^7 is with electrocompetent bugs. With that I've had up to 10^8 and even
The method described in Inoue, Nojima and Okayama, Gene, 96, 23-28 (1990) does
yield 10E8 consistently and doesn't require any special equipment, except
perhaps a refrigerator incubator. The authors observed that growing the E.
coli cells at 18C prior to the washing steps increased the transformation
efficiency and also made less critical the OD at which one harvested the cells.
Growing the cells at room temperature gave results between 37C and 18C growth.
You should be able to find prior bionet.molbio.methds-reagnts discussion on
this method by Gopher searching the archives at net.bio.net.
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email: tan at aeolus.vmsmail.ethz.ch
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