digesting the ends of PCR products

Martin Hughes mhughes at fhs.csu.McMaster.CA
Sat Feb 1 09:02:43 EST 1997


On 31 Jan 1997, it was written:

>    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?

Use an amplification enzyme with exonuclease activity, or treat your PCR 
product with Klenow to get blunt ends.
Ligate your product to form concatamers (so you don't have to worry about 
the number of bases at the end), and then digest with KpnI followed by BamHI.
You should be able to see the difference between your ligated and KpnI cut 
products on a gel.

Cheers

Martin Hughes
Department of Biochemistry
McMaster University
Hamilton, ONTARIO, Canada.




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