Ligations. Restriction of PCR products with sites in primers.

Brain Foley brianf at dna.uvm.edu
Fri Apr 9 17:57:29 EST 1993


I have one more tip for ligationg PCR products:

People often have difficulty cutting a PCR product when the
restriction site is built into the primer, so that the site is on the
very end of the molecule.  Current Protocols recommends ligating the
PCR product into concatamers, then cutting.  But the product needs to
be blunt-ended first with klenow (to fill in those pesky TAQ
overhangs) and then blunt ended ligations are not efficient.

My tip is to use terminal transferase to add a nonspecific 3' overhang
of 50 to 300 bases.  Although this is single-stranded, it seems to
help the restriction enzyme cut the site that is now near the ss-ds
boundary.  A second advantage is that you can SEE if the PCR product
is cut.  The product will be a smear on a gel after the terminal
transferase has added variable numbers of bases.  If your enzyme cuts
well after this treatment, you will again get a nice sharp band.

Even if your enzyme does not cut 100%, you can gel-isolate the
molecules that were cut.  If your enzyme does not cut, you can SEE
that without having to do a ligation/transformation.
--
********************************************************************
*  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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