digesting the ends of PCR products

Fri Jan 31 14:08:01 EST 1997

@bham.ac.uk (s j peake) writes: >    I've placed restriction sites in the ends of my PCR product by engineering 
> the sites into my primers.  The BamH1 site at one end cuts okay, but the Kpn1 
> site at the other end doesn't cut at all . Both sites have two base pairs to 
> act as a clamp which according to the NEB catalogue should be fine.  Has 
> anyone got any ideas or do l need to get a new primer?

Another way around this problem is to ligate your PCR product into a T/A vector.
This not only supplies the neccessary bases for your enzyme to clamp on to, but also 
results in your insert being hard copied into a vector and bug so you'll always 
have a stock stored in the freezer.  Another problem could be the primer itself.
I have seen many primers with sequence errors.  The only way I know of to check 
for sequence errors is sequencing.

Hope this helps, Mike.

Michael E. DeGraaf
Pharmacia and Upjohn Inc.

MEDEGRAA at am.pnu.com

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