digesting the ends of PCR products

Bernard Murray bernard at elsie.nci.nih.gov
Fri Jan 31 18:39:55 EST 1997


In article <5ct7hl$gn1 at usenet.bham.ac.uk>, @bham.ac.uk says...
>
>   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?

My bid would be to avoid KpnI (ack!).  Yes, it does work but can be a
bit fussy.  You might try swapping to Acc65I as touted by the people
at Promega.  I've done this and found it a more tolerant enzyme but
there are two gotchas;
1) It is a *neo*schizomer not an isoschizomer so you have to cut both
oligo and vector with this enzyme.
2) It is susceptible to Dcm methylation - check the context.

The invaluable NEB chart claims 99% efficiency for Acc65I when cutting
two bases from the end so this sounds as if it would work for you.
	If you are staying with KpnI then run the reaction in a
relatively large volume to dilute the glycerol to below the star
activity concentration.
	I hope something works,
		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)




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