Adding Restriction sites to Primers

[Brian Foley]

BMA14432 at vax1.utulsa.edu wrote:
: Hello netters,
: I've picked a couple of primers to PCR my target and now wouuld like to
: add restriction sites to the 5' ends of them. What are the
: pitfalls, if any, to be avoided in doing this. Any helpful hints or
: anecdotes will be greatly appreciated! Thanks in advance

	This has been discussed a LOT before, so it would be worth it for 
you to check the archives of this group (via GOPHER to Indiana:IUBIO is one 
way to do so).  Briefly, the only probelm to avoid is making usre that 
the restriction enzyme you choose is able to cut very near the end of a 
ds-DNA molecule.  I use BamHI and EcoRI with only 3 bp extensions off the 
end after the recognition site, and they work fine.  Some enzymes will 
not cut unless the site is a fair distance from the end of a molecule.

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