PCR cloning

David Clayton dclayton at uiuc.edu
Tue Jun 27 17:32:33 EST 1995


In article <3snto9$56s at cc-server9.massey.ac.nz>, "R.D.Johnson"
<R.D.Johnson at massey.ac.nz> wrote:

> I found that performing a  self ligation after T-tailing the vector and 
> then cutting out the linear form from a gel dramatically improved my
> cloning efficiency, presumably because non T-tailed vector is removed.
> 
> Rich

Keep in mind that the non T-tailed vector will not be completely
'removed', but will lie in a supercoiled pattern on the gel; you will
*reduce* the presence of non T-tailed vector in your ligation, but you
will not eliminate it completely by this method.

If the vector is *phosphatased* prior to ligation, blunt-ended vectors
should not self-ligate, while successfully T-tailed vectors should happily
accept a PCR product.

Rick



More information about the Methods mailing list