Why use Klenow on a PCR product to clone?

Brain Foley brianf at med.uvm.edu
Tue Nov 9 16:38:35 EST 1993


Dear BioNetters:

	I was recently asked why so many of the protocols people have 
supplied to this list for cloning a PCR product include treating
the PCR product with Klenow fragment to "clean up" the PCR
product ends prior to blunt-end ligation.

	The PCR product has a 3' overhang, usually an A, added by the
polymerase.  Klenow lacks 3'-5' exonuclease activity, and is only
a 5'-3' polymerase (unless I'm mistaken).  Thus it does not seem
to us that Klenow should actaully do anything to "clean up" the
3' overhanging A.

	Are we missing something???

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *



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