hwong at acs.ucalgary.ca (Henry Wong) wrote:
>Getting no ligation whatsoever with different ligases from different
>companies (BRL, Promega, Boehringer, Pharmacia), gel purified (Sigma low
>melt), single cut vector religating on itself, blunt (A-overhangs into
>T-vectors) and sticky end ligations,addition of ATP into ligation
>reactions, ligating at 12 degree and 15 as well. What to do, any
>suggestions? Please help. Oh yes, cells are competent.
>Henry Wong
>MID, Universtiy of Calgary
>email: hwong at acs.ucalgary.ca
It is possible you're getting inhibition of ligation. Ligase is
sensitive to ammonium salt, high concentration of sodium chloride
and a few other things I don't recall right now (I'm writing from
home, so I can't check). Some grades of agarose are inhibitory, too,
and, I think, so is TBE electrophoresis buffer. Check with your
supplier. They should have a list of what to watch out for when doing
ligations.
Good luck,
Danuta
