digesting the ends of PCR products
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.
>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).
Program in Immunology
Washington University - St Louis
brett at borcim.wustl.edu
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