Adding Restriction sites to Primers

micprf at lure.latrobe.edu.au micprf at lure.latrobe.edu.au
Thu Mar 24 04:26:48 EST 1994


In article <940309084116.2401704d at vax1.utulsa.edu>, BMA14432 at vax1.utulsa.edu 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
> BMA14432 at VAX1.UTULSA.EDU
> 
> All views are MINE.

We just went through a deal of drama using two different restriction sites
and a 2 base-pair GC clamp with enzymes that according to the catalog info
should have worked under these conditions. Our solution was to choose a
strategy that gave us multipe chances:
We incorporated in the 5' ends
a. the sites we really needed (this was an expression cloning)
b. then outside that an EcoRI site for in each primer (gives extra
bases as well as an extra cut site, and in our experience EcoRI cuts well
at ends)
c. then the usual GC clamps
d. the primers were phosphorylated

Becuase the primers were phosphorylated we could concatemerize them
by ligation of the gel purified PCR product, before cutting.
Because of the EcoRI sites we could clone first using them and then
subsequently cut out the insert using the two enzymes we really needed
for cloning into our expression site in frame.
Alternatively we could clone directly from the concatemerized
products using these 2 enzymes rather than EcoRI.
Finally we could use the 2 desired enzymes without concatemerization,
relying on the extra bases in the EcoRI site+GC clamp to give enough
space.
In this way we had several alternatives for cloning, so that if one
approach did not work, another might with the same PCR product!
Becuase of our earlier difficulties, we tried first with EcoRI
and concatemerized product and it worked... so we haven't tested the
other possibilities.
                          Hope this helps,
                          Paul Fisher (Microbiology, La Trobe Uni).




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