fill-in and blunt-end ligation

Ian A. York iayork at panix.com
Fri May 23 07:52:10 EST 1997


In article <5m01ma$dkq$1 at miranda.its.deakin.edu.au>,
Karthi Ramachandran <Mari.L.Shinohara at dartmouth.edu> wrote:
>
>I do my sticky end ligs at Room T for 1 to 2 hrs and Blunt end ligs over night 
>at 16 degrees C. (I have a water bath set up at 16 degrees in out 4 degree 

I strongly suspect that most protocols for ligation are massive overkill. 
New England Biolabs catalogue has a figure showing that their ligase leads
to near-complete ligation of sticky ends within 10 minutes (room
temperature) and extensive ligation of blunt ends within 30 minutes at
room temperature.  (I'm awfully suspicious about the "fast ligation"  kit,
which supposedly has some magic ingredients in the buffer or something;
its claims actually are not significantly better than standard ligase will
do.)  I've had no problems with 1 hour ligations, although I usually do
leave them overnight just for convenience.

Ian

-- 
      Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
      "-but as he was a York, I am rather inclined to suppose him a
       very respectable Man." -Jane Austen, The History of England



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