ligation problems

Karl Voss karl at
Fri Sep 22 10:58:24 EST 1995

> In article <1995Sep21.101757.1 at>, egorham at wrote:

> > We are experiencing difficulties ligating a 600 bp PCR product into the
> > pTrx-fus expression vector.  We designed PCR primers to incorporate BamH1 and
> > Kpn1 restriction sites for directional cloning.  We have gel purified our
> > insert using ulta-pure Sea Plaque agarose.  The vector has been treated with
> > Alkaline phosphatase to remove 5' phosphates and inhibit self-ligation of the
> > vector.  Our controls show the enzymes are cutting and that the transformation
> > is efficient.  Any suggestions regarding improved ligation efficiencies would
> > be greatly appreciated.-- 

When I first tried this method I never got good results.  I think that my 
problem was that I was over digesting the PCR product and mangling the ends.

I started getting much better results when I used only 5 units of restriction
enzyme and digested for one hour only.

What I am having trouble with now is cloning PCR products by blunt ending.
If anyone has a protocol that they have found works well I would love to 
hear it



Karl Voss
Department of Chemistry
University of Alberta
Edmonton, Alberta, Canada
T6G 2G2

karl at
phone 403-492-0222
fax   403-492-8231

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