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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