In <stephan.witte-041295105947 at marvin.biologie.uni-konstanz.de> stephan.witte at uni-konstanz.de writes:
> I«m looking for highly competent E.coli cells for a library transformation.
> Our homemade CaCl2-competent cells yield app. 5E4 transformants per ug
> DNA (6 kb plasmid)
> I think, this is not much.
> Are there better protocols for preparing highly competent coli-cells?
> What to think about Stratagenes ultracompetent cells? What«s the trick?
I contacted Sratagene on that very point over a year ago. Their reply was
that thier protocol for peparing the ultracomps was nothing more than a
modified form of the Hanahan protocol. However, these modifications were
proprietary, of course, and would not be revealed - otherwise they'd loose
thier market share! With standard CaCl2, I've managed only as high as 10^4
cfu/ug. With Hanahan and Inuoue procedures, I consistently get into the
10^6 range and have, from batch to batch, flirted with low 10^7. For me,
this is more than adequate for standard ligations but we do keep ultracomps
around for libraries and large insert ligations (>8kb inserts). BTW - the
cells that I've used in these protocols are Stratagenes XL-2 mrf'.
I'd love to find a chemical protocol that would get me 10^8 on a regular
basis. I should note that 10^8 is all I've ever managed to get with
electroportation as well.
Hope this helps,