digesting the ends of PCR products

Alan Lau AL18 at le.ac.uk
Fri Jan 31 12:22:55 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?

What you can do is make DNA concatermers of your PCR products.  This way
your KpnI 
sites become internal and not at the ends and eliminates the problem of
inefficient cutting.  
Clean up your PCR products as normal then incubate them for 2 hours at
room temperature in 
a standard Klenow polymerase fill-in reaction but also add T4 kinase and
T4 ligase enzymes.  
I have done this in the past to produce end-to-end DNA fragment
concatermers which then cuts 
efficiently.

I hope this helps

Alan Lau

-- 
al18 at le.ac.uk
Dept. Microbiology and Immunology
University of Leicester. UK.
LE1 9HN



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