restriction digestion of PCR product for cloning

Deb Britt Deborah_Britt at brown.edu
Wed Oct 8 14:38:10 EST 1997


I routinely cut PCR products that have three bases tacked on past the
restriction site in the primer, although cutting efficiency depends on the
enzyme you are using (check the NEB catalog, as suggested by others).  I
either gel purify the PCR product or, if it's clean, just
phenol/chloroform treat and run it through a Pharmacia HR400 column (or
you can EtOH ppt).  I set up the digest with excess enzyme and digest for
long periods (6-8 hrs, sometimes overnight), then ligate as usual.  I
screen the colonies by PCR, and even if I have high background of colonies
on the vector-only plate I nearly always turn up at least a couple of the
clones I want.

Deb Britt

In article <Pine.GSO.3.96.971002173330.27352A-100000 at cougar.vut.edu.au>,
s9401501 at COUGAR.VUT.EDU.AU (Matthew Knight) wrote:

> I am having problems digesting a PCR product which has restriction enzyme
> sites placed into the PCR primers, thus they are very close to the end of
> the PCR product.
> 
> Does anyone have any suggestions on how I can be sure that the
> restriction enzyme is going to cut the PCR product. And will the
> restriction enzyme cut with the sequence being so close to the end of the
> product (i.e. about 5 or 6 bases)
> 
> Thanks in advance
> 
> Matthew Knight

-- 
Deborah Britt, Ph.D.
Department of Medical Oncology
Rhode Island Hospital




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