Electrotransformation efficiency

ladasky at my-deja.com ladasky at my-deja.com
Fri Jul 14 05:37:44 EST 2000


In article <8kmn5a$5ca$1 at balder.adm.ku.dk>,
  "Jens Mollerup" <jmollerup at mcb.molbio.ku.dk> wrote:
> Hi Scientists
> When we do electrotransformation of E.coli (SURE or JM109 cells) we
> get a low tranformation efficiency (3 x 10E5) which certainly is
> lower than could be expected!
> Competent cells:
> The cells are harvested at OD550 0.8, washed twice in milliQ water +10%
> glycerol (kept at 4C all the time) and frozen in liquid nitrogen,
> stored at  -80C.
> Transformation:
> Invitrogen 0.2cm cuvettes, BioRad Gene Pulser II at 2.5 kV, 200 ohm,
> 25 microFahrad, 80 microL cells + 1 microL DNA - low salt (e.g., pBD-
> GAL4), E. coli and cuvettes are kept on ice! Then growth at 37C in NY
> for 1hr before plating to ampicillin plates.
>
> We have tried several times with different E.coli (and preparations
> thereof) but the transformation efficiency is low and useless. Does
> anyone have an opinion of what to do or what we do wrong?

I had a problem like this.  It plagued me for a month.  I got good
transformation efficiency with uncut plasmid, but terrible efficiencies
with the ligation mix.  I ordered a completely fresh cloning kit and had
the same problem.  (You did not mention whether you performed a no-
insert, intact plasmid control.)

Finally I decided to question our lab's sterile water, which came from
our then-new MilliQ filtration system (followed by autoclaving, of
course).  I got some sterile distilled water from another lab and my
ligations gave great results.  More recently, a fresh preparation of
water from our lab's MilliQ system worked fine.  I have no idea why the
ligation reaction was unhappy with our lab's water.  I had been using
this same water for PCR with no troubles.

--
John J. Ladasky Jr., Ph.D.
Department of Structural Biology
Stanford University Medical Center
Stanford, CA 94305
Secretary, Californians for Renewable Energy <http://www.calfree.com>


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