Adding Restriction sites to Primers

Dr. Robert Darnell darnelr at rockvax.rockefeller.edu
Fri Mar 11 07:45:14 EST 1994


In article <wgallin.1113622918C at NEWS.SRV.UALBERTA.CA>,
wgallin at gpu.srv.ualberta.ca (Warren Gallin) wrote:

> 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

> >Mark Brudnak

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

We have also used the Invitrogen kit successfully, but are intimidated by
the high cost of the kit, the key ingredient of which is the vector.  In
follow up to your comment, is there a straightforward way to prepare a
vector with a single 3' overhanging T?  Looking through the NEB catalogue,
it is not obvious which 5/7bp cutter could do this.  Or is there a simpler
way...?



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