digesting the ends of PCR products

brett brett at BORCIM.WUSTL.EDU
Fri Jan 31 18:00:14 EST 1997

>s j peake wrote:
>>    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?
>A workaround to this problem would be to self-ligate the PCR product
>before digestion, that way if more bases are required at the KpnI end
>they would be provided by the next copy of the PCR product.
>Hope this helps.
>Darren Tyson
>Ph.D. Candidate in Cell and Molecular Biology
>Saint Louis University
>tysondr at slu.edu or darren at primary.net

Agreed, but remember to kinase your oligos before PCRing (saves a clean up
step to get rid of NH3 in many PCR buffers).

Brett Lindenbach
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

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