Competent Cells - making the best

tfitzwater at NEXSTAR.COM tfitzwater at NEXSTAR.COM
Mon Feb 22 21:03:32 EST 1999



I prepare electro-competent DH5alpha cells with transformation efficiencies of
1.2e10 cfu/ug
by following (with one modification) a protocol by William J. Dower that was
published in a Bio-Rad newsletter.
The citation in that article was a paper by Dower et. al. Nuc. Acids Res.
16:6127 (1988).
The change I made resulted in better transformation efficiency in my hands:  all
 of the water and 10% glycerol
that is used to prepare the cells is filter sterilized, NOT autoclaved.

Tim Fitzwater
NeXstar Pharmaceuticals

In article <w.ingram-2202991921470001 at b-wainwright.cmcb.uq.edu.au>,
w.ingram at cmcb.uq.edu.au (Wendy Ingram) wrote:

> I'm sure this question has been posed in this newsgoup before ... but what
> is the best way of making homemade competent cells??? (or who sells good
> commercial ones that don't cost the earth and work reliably...)
> In our lab we have tried various protocols and several E.coli strains over
> the last couple of years, but never seem to get a transformation
> efficiency better than about 10 to the 6. Does anyone out there have a
> never fail super good protocol they would like to share? What sort of
> efficiencies to other people out there really get???






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