Ligation Help Requested

Zophonias Oddur Jonsson zjons at rhi.hi.is
Sun Mar 28 20:45:29 EST 1993


In <27MAR93.19921652.0075 at UNBVM1.CSD.UNB.CA> E85J at UNB.CA (E85J000) writes:

>Dear peoples:

>I'm in serious need of some help!  I have a PCR product with
>engineered NdeI cut sites at both 5' & 3' termini.  I'm trying,
>without any success, to cut and ligate into the NdeI site of
>pET-11a. Etc....

I recognize your problems.
I had a similar experience when PCR-cloning with an engineered NdeI
site.  Then I came across an article where it was suggested treating the
PCR product with proteinase K (and then phenol-extracting carefully of
course) before digesting with restriction enzimes.  Unfortunately I can't
find the article at the moment so I can't give you the reference, but 
anyway this worked out fine for me and I got the desired ligation.

I hope this information is of some use !

Zophonias O. Jonsson: zjons at rhi.hi.is
Department of Molecular Genetics,
University of Iceland, Reykjavik, Iceland.



More information about the Methods mailing list