Adding Restriction sites to Primers

Warren Gallin wgallin at gpu.srv.ualberta.ca
Wed Mar 9 12:47:58 EST 1994


In Article <940309084116.2401704d at vax1.utulsa.edu>, 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
>
>Mark Brudnak
>University of Tulsa
>BMA14432 at VAX1.UTULSA.EDU

Two things that we have noticed. 1) Even with GC clamping, we have had a lot
of trouble using sites on the end to subclone.  We switched to A-T
subcloning using the Invitrogen kit and got beautiful results (I hae no ties
to the company, and we're currently making some home-made vector to avoid
the high cost).  2) When we compared the primers with and without the
restriction sites added, we did not get the same band patterns; overlap but
not identity.  This was using fairly degenerate primers at low stringency.
   Adding sites to the end may not be necessary, depending on what you are
doing, so you might want to consider alternatives.  Not that they don't
work, but they are also not problem-free.

Warren Gallin,
Department of Zoology, University of Alberta
wgallin at gpu.srv.ualberta.ca



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