Blunt end ligations

malaine at sara.cc.utu.fi malaine at sara.cc.utu.fi
Tue Feb 8 12:22:45 EST 1994


Hi netters!

Few months ago we had a discussion here about _blunt end_ ligations and now I
would like to extent this conversation a little bit. I would like to know how
you blunt the cohesive ends -It's not always that easy :-) :-)

We usually fill 5'-overhangs using Klenow and dNTPs, and it works well. But
3'-overhangs seem to be more difficult (=efficiency is not that great). We have
used Klenow without dNTPs to chew the 3'-overhangs (about 20 min at RT) and
then added the dNTPs, but after that ligations haven't worked well. Instead
somebody adviced us to use T4 DNA Polymerase to get rid of 3'-overhangs, but we
haven't tried it yet.

Now I would like to know how you usually perform these reactions at the way
that they work? I would appreciate any good advice or hint to make these simple
things to work...

Thanks, 
Marko Laine


Marko Laine
Univ. of Turku 				Malaine at sara.cc.utu.fi
Dept. of Plant Physiology               Malaine at polaris.utu.fi
Biocity, 6th floor
20520 Turku
Finland



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