digesting the ends of PCR products
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
> the sites into my primers. The BamH1 site at one end cuts okay, but the
> site at the other end doesn't cut at all . Both sites have two base pairs
> 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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