Help with ligations

Danuta Bois dbois at netsrq.com
Thu Jun 13 17:48:02 EST 1996


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




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