Blunt end ligations

Brian T. Greuel greuelb1 at jaguar.uofs.edu
Tue Feb 8 14:12:10 EST 1994


In article <1994Feb8.192245.1 at sara.cc.utu.fi>, malaine at sara.cc.utu.fi writes:
> 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, the standard approach for 3' overhangs is just what you mentioned:  
chew back with T4 DNA polymerase in the presence of dNTPs.  It works well in 
my hands...

Brian Greuel
University of Scranton
Scranton, PA







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