digesting the ends of PCR products

AMICVS CONSTANTINI COMES at NOVAROMA.GOV
Wed Feb 5 20:50:56 EST 1997


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

I used KpnI all through graduate school for my transformation constructs.  I
found that cutting with a high salt enzyme first would kill KpnI activity,
even if phenol-chlorof. and EtOH ppt. intervened.  presumably enough salt
makes it through to kill the KpnI.  If I used KpnI *first*, then used e.g.
BamHI I had no problem.  



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