3' overhang ligation

Vladimir F. Yamshchikov vlad at vlad.microbio.emory.edu
Tue Aug 24 00:23:28 EST 1993


In article <1993Aug23.141601.1 at hkucc.hku.hk>, lhyatt at hkucc.hku.hk wrote:


> several times, with phosphatased vector, and always with the same result: all
> colonies are religated vector (or linear vector, if my concentration of DNA is
> too high).

 Linearized plasmids from E.coli?!

> but i have lost it. Is there a special trick to these ligations? I have also
> tried doing the same insert and vector with 5' overhang (Asp718), but the
> enzyme does not give good digests in my hands (I have tried 2 batches, and the

> I would be very grateful for any advice, experience of similar problems,
> attempts to use Asp718 and isozymes etc.

I did not have any problems with Asp718. A very nice enzyme. Never
experienced
any problems with 3'-ligation. How did you isolate your DNA?  At first
glance,
but I may be wrong, there seems to be a problem with DNA quality.

> Lucy

Sincerely, Vladimir.



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