problem with ligation of linker in vector

Christian Ebeling cebelin at gwdg.de
Wed Sep 17 15:16:11 EST 1997


Koen De Smet wrote:
> 
> bvb wrote:
> >
> > Fellow netters,
> >
> > I've some difficulty to ligate (sticky) a 27 bp linker in my vector.
> >
> > I've cut my vector with Xho I and Spe I (only sites in "multiple cloning
> > site") and purified the cut vector with the Qiaquick kit from Qiagen.
> > For ligation of different fragments (which have Bam HI and Not I ends)
> > in this vector I want to create a Bam and a Not site by ligating a
> > synthetic linker with internal Bam and Not sites in the Xho/Spe site of
> > the vector. 

> I once had a similar problem, cloning in a vector cut with BamHI/ClaI.
> It should have worked but it didn't. Eventually I kinased the oligos and
> added this to the ligation. It worked, but don't ask me why!
> 
> I thought that kinasing the oligos may lead to the introduction of long
> repeats of oligos in the vector, but when I sequenced one, there was
> only one linker in it; just what I wanted.
> 
> Koen De Smet

I dont understand the problem to have repeats in the site. If you have
repaets in your site cut it with BamHI or NotI, seperate the
vector-fragment on a agarose gel and  and religated it.
Christian



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