Ultracompetent E.coli cells?
e-anderson at ski.mskcc.org
Sat Dec 9 16:04:21 EST 1995
In article <4aat3b$j5n at epx.cis.umn.edu>, shin at biosci.cbs.umn.edu (Shin
> David L. Haviland, Ph.D. (HAVILAND at KIDS.WUSTL.EDU) wrote:
> : 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
> : 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.
for what it's worth, i tried the Chung, et al "TSS" protocol (PNAS, 1989,
V. 86, 2172-2175) that Paul Hengen mentioned in this thread the other day
with JM109s and while i haven't quantitated them yet, got amazing results
with a TA-PCR ligation compared to a side-by-side with CaCl2 JM109 that i
made about a week ago. an eyeball estimate is that i got about 50-100x
more colonies with the TSS bugs than with the CaCl2 bugs. i'm going to do
a pUC18 control next week and will post results if anyone cares. the best
part about the protocol is how quick and easy it is compared to the
Maniatis CaCl2 protocol. it took me about 20 minutes from bugs in the
37oC shaker to transformation.
p.s. David, say hi to Mike Crossman for me...we used to be in the same
lab at WUMS.
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