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Ultracompetent E.coli cells?

David L. Haviland, Ph.D. HAVILAND at KIDS.WUSTL.EDU
Fri Dec 8 12:39:32 EST 1995


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?

Stephan

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,
David




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