Ultracompetent E.coli cells?

Paul N Hengen pnh at cockleberry.ncifcrf.gov
Mon Dec 11 15:06:32 EST 1995


David Micklem (drm21 at mole.bio.cam.ac.uk) wrote:

| Inoue, H. Nojima, H. and Okkayama H.: Gene, 96 (1990) 23-27
| Works very well.

: Sure does!  Still, if anyone has compared the cells from this protocol
: with the ones from the protocol Paul Hengen posted a few days ago then I'd
: love to know.  This seems to be unbelievably simple - maybe I should try
: it next time I run out...  Does freezing affect the competence of these
: cells Paul?

In that article (Chung1989), JM109 cells were frozen at -70C in TSS (LB + 10%
PEG, 5% DMSO, and 20-50 mM Mg2+, pH 6.5). Fresh unfrozen cells gave 5.12x10^7
and frozen cells 1-18 weeks later gave 6.17x10^7 transformants per ug of pUC19
DNA. Epicentre claims the standard TSS gives 2.4x10^6 and their modified TSS
protocol (only scaled up as far as I can tell) gives 7.5x10^6 HB101
transformants per ug of pGEX-2T DNA using 5ng.

On the other hand, Inoue1990 report 1.44x10^9 JM109, 1.84x10^9 DH5, and
1.00x10^9 HB101 transformants per ug pBR322 DNA.  Still I'm always doubtful of
efficiencies reported because they always seem too high compared to "real"
everyday results. Just look at the results of Epicentre...you'd think if they
got great results, they would advertise this...Anyone else notice this?
I think it is more strain dependent or maybe the tubes used (glass versus
polypropylene), handling, etc., than anything and I always have to try any
protocol myself to be sure things are on the up-and-up. If you give it a try,
please let us know what happens.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
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