digesting the ends of PCR products

David D Morgan d.d.morgan at ncl.ac.uk
Wed Feb 5 12:03:02 EST 1997


On Wed, 05 Feb 1997 17:50:56 -0800, "AMICVS CONSTANTINI"
<COMES at NOVAROMA.GOV> wrote:

>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.  


I read somewhere that digestion could be improved by first
self-ligating the pruified PCR product before performing the
restriction digest but I haven't tried it myself !

Hope this helps
Dave Morgan

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