PCR product cloning

eric anderson e-anderson at ski.mskcc.org.nospam
Thu Apr 24 11:09:58 EST 1997


In article <5jisc3$ofk at info.abdn.ac.uk>, mbi071 at sysc.abdn.ac.uk
(j.r.stephen) wrote:

> David Neece (dneece at ux1.cso.uiuc.edu) wrote:
> : I have also had a lot of success with Promega's pGEM-T kit.  In my
experience, 
> : two things will improve cloning efficiency:
> 
> : 1) Clean up your PCR product before ligating it into the vector (I use
the Qiex 
> : gel purification method).
> 
> : 2) Use high efficiency bugs for the transformation.
> 
> : For me, using "subcloning efficiency" heat-shockable E coli usually
resulted in 
> : about 5 - 10 white colonies per plate.  Then I switched to
electroporation for 
> : the transformations, and now get 100's of whites per plate -- most of
which are 
> : positive for my insert.  If you don't have access to an
electroporator, high 
> : efficiency heat-shockable E coli should improve your results.
> 
> : Hope this helps,
> : dAVE
> 
> I would agree here, don't think there is really any point in trying to clone
> into low-efficiency E. coli, I always buy super-competent cells from
Stratagene
> (XL1 blue Kan-resistant strain) for cloning T-vector libraries.
Expensive, yes,
> but worth it!
> 
> Good luck,
> 
> John Stephen

just thought that i'd offer my 2 cents on this subject.

i made a TA-vector using an  oligo that i previously mentioned on the
group, use 2uL of a PCR reaction straight out of the tube (less if the
reaction was incredibly efficient and a little more if there's not too
much product) and use homemade CaCl2 DH5alphaF' cells.

out of this kind of TA subcloning i usually get 20-40 white colonies with
a 3 or 4:1, white:blue ratio.  this costs essentially nothing except for
the ~$65 we spent on making the oligos to make the original vector
(subcloned into Bluescript) and the cost of the enzyme to cut our vector
and the ligase.

YMMV of course, but this seems to work well for me and the couple of
people in the lab who also use it.

eric

-- 
Eric C. Anderson
Memorial Sloan-Kettering Cancer Center
Sloan-Kettering Institute
1275 York Ave. Box 470
New York, NY  10021
(212) 639-2977
e-anderson at ski.mskcc.org
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