Adding restriction sites to PCR primers

D.D. Morgan D.D.Morgan at ncl.ac.uk
Tue May 16 09:05:38 EST 2000


Susanne Rohrer ("srohrer(removethis)"@immv.unizh.ch) wrote:


> Beverly Metcalfe wrote:

> > I am designing primers and I want to add restriction enzyme sites to the
> > 5'ends.  To do this, do I just add the restriction site to the 5' end, or do
> > I need to add extra bases after the enzyme's site so the the enzyme will be
> > able to cleave it?

> depends on the enzyme - look it up in the back of the New England Biolabs
> Catalogue, or  get the same information on their website (www.neb.com).  Some
> will not cut very well.  For digestion of PCR ends, I recomment you do it
> overnight in an incubator.

Alternatively, just add the restiction site to the end of the primer with
no extra bases and then clone the PCR product into a T-tailed vector
before cutting out and cloning in your final vector - I do this routinely,
it only takes a day or two more to do and there's no doubt about the
enzymes cutting efficiently

Dave Morgan
--

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