what's the best way to clone PCR products?

Mike Preston preston at warren.med.harvard.edu
Tue Sep 27 12:50:16 EST 1994


some material deleted.
 GeneClean should effectively remove any detergent
> >or salts present in the PCR buffer, so digestion of the PCR product should
> >not be inefficient if the conditions of the digest are correct. Good luck.
> 
> >Tracy Aquilla, Ph.D.
> >Molecular Physiology and Biophysics
> >University of Vermont
> >aquilla at salus.med.uvm.edu
> 
> Well I tried cloning 1 lousy PCR product with restriction sites (Eco RI and 
> Hind III) for over 4 months and never got close.  Some PCR products just will 
> not cut with enzymes, 8 base overhang or no.
> 
> I routinely use Promega's PGEM-T  with electroporation now and never have any 
> problems
> ------------------------------------------------------------------------------
> Dr Mike Poidinger        
> Microbiology, UWA       ==> Animal Welfare NOT Animal Rights       
> Australia                        ==> Sonic the Hedgehog says "It's your cone"
> mikep at uniwa.uwa.edu.au 
> -----------------------------------------------------------------------------

(Some material deleted)

	I have the feeling that under the appropriate conditions for the
restriction enzymes and a reasonable number of "protecting" bases at the
ends of the sites in the primers, that most enzymes will cut PCR products. 
The problem may lie in getting rid of all of the Taq.  If there is residual
Taq and dNTP's in the digest, your overhangs may fill in, even one base may
interfere with the ligation.
	It sounds as if Tracy has the right idea in using gene clean to purify the
PCR product first.

	For those of you who have had success direct cloning of PCR products do
you have a step that gets rid of the Taq?  Conversely, those of you who are
having trouble, do you get rid of the Taq?

 
Mike Preston
Channing Laboratory
Brigham and Women's Hospital and Harvard Medical School
preston at warren.med.harvard.edu



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