digesting the ends of PCR products

Ashok Aiyar aiyar at ebv.oncology.wisc.edu
Mon Feb 3 17:41:27 EST 1997

On 31 Jan 1997 16:44:37 GMT, s j peake <@bham.ac.uk> 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?

Either phosphorylate your oligos before the PCR (preferred), or 
phosphorylate the purified PCR product.  Then self-ligate it.  
Restriction digest the multimerized product.

This will work as long as a significant proportion of your PCR
product is blunt-ended.  If this is not the case, you should blunt
the ends using T4 DNA polymerase prior to the self-ligation.

Ashok Aiyar, Ph.D.
McArdle Laboratory for Cancer Research
aiyar at ebv.oncology.wisc.edu

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