Cloning in pGEM-T

Bernard Murray, PhD spam at 127.0.0.1
Wed Nov 24 20:14:36 EST 1999


In article <81hpbe$r7k$1 at feresa.bio.vu.nl>, "Neil Saunders"
<saunders at bio.vu.nl> wrote:

> In my experience with pGEM-T, PCR product purification is not necessary if
> you have a nice clean, specific PCR reaction (one major band on the gel).
> If you clone direct from the reaction mix, you sometimes get one or two
> contaminant inserts, but these occur at much lower frequency than the main
> product and of course are easily distinguished on digestion

> Christophe Jacob <jacob at scbiol.u-nancy.fr> wrote in message
> news:jacob-2411991849160001 at betula.scbiol.uhp-nancy.fr...
> > Does anyone know if the purification of PCR fragments (from agarose gel or
> > with a column to eliminate other bands and/or oligonucleotides) before
> > cloning in T/A vectors is a critical step ??

Running the reaction on eg. a G25 column takes only a short amount
of time and may help the ligation efficiency (and is a quick
salve to the conscience).  As Neil points out, this still only works
with a relatively clean PCR giving a single product (otherwise
purification on an agarose gel would be a good idea.

   Bernard

-- 
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF




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