Adding Restriction sites to Primers

Christopher Cilley cdcilley at
Sun Mar 27 14:24:39 EST 1994

In article <940309084116.2401704d at>, BMA14432 at writes:
> 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

I follow the guidelines in the appendix for the NEB catalog listing cutting
efficiencies vs. overhang length.  Whatever the longest they report, I add a
couple of extra bases onto that.  I haven't had problems with the length of my
5' overhang.  I was able to get a primer with a 77 base overhang to PCR up
just fine (the homologous region was about 22nt and I annealed at 55oC).
Unless there are some constraints on cost, don't be stingy with the length
of the overhang.

	- Christopher

"People who don't fight the unjust condemnation of others 
 but internalize it are, Miss Manners admits, badly in need of 
 lessons in self-esteem."	-Judith Martin (aka Miss Manners)

cdcilley at

	- Christopher

"People who don't fight the unjust condemnation of others 

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