PCR cloning

eric anderson e-anderson at ski.mskcc.org
Fri Mar 28 10:09:41 EST 1997


In article <EnCj7Bu00iWl09Q3w0 at andrew.cmu.edu>, Monica Ruiz-Noriega
<mr6h+ at andrew.cmu.edu> wrote:

> Recently I've been trying to clone a PCR product but my attempts haven't
> been succeful. Do I need to keep anything in mind when doing so?.
> What I did: PCR out a 2.1 kb fragment with SmaI and Apa I sites at the
> ends. Phenol-chlorophorm, ethanol precipitate my fragment and cut wiht
> the enzymes mentioned above. Then I just tried to ligate this product to
> a linearized plasmid cut with the same enzymes. Before trying the
> ligation reactions I checked both DNAs by electrophoresis and looked
> fine (not degraded and in the case of the plasmid, looks cut)
> I cannot get any E. coli transformants. It is the first time that I
> subclone PCR products and I do not know if I need to do anything
> different. The ligation reactions that I am using have always worked
> before but they're not working now. 
> Any suggestions and comments will be highly appreciated.

in my experience, cutting PCR products with enzymes that have sites
designed into the primers (with or without all the extra bases that are
needed to flank it) rarely works.  you have a few options:

1.  blow some money on a commercial TA cloning system (Invitrogen, Promega
and Stratagene all make kits that work decently to without fail).  this is
the quickest and easiest way to go.

2.  make your own TA vector.  if you can get oligos very cheaply (we have
a machine on the floor here that we use to make them ourselves for about
50 cents/base) you can design a pair that incorporate sites that, when cut
will leave the T-overhang you need.  if you intend to do a great deal of
PCR subcloning in the lab, this is an excellent investment.  if you just
need this one clone then this is a waste of time (a week or so to do all
the synthesis, cloning and screening) and money.

3.  gel purify your fragments after PCR, fill them in with Klenow to blunt
them and then drop them in BlueScript or pUC or something in a blunt site,
then cut them out with the sites you designed into your primers.

good luck,

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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