Blunt end ligations - another question

[Brian T. Greuel]

In article <2jb6m2$t6m at mserv1.dl.ac.uk>, Helge Weissig <helgew at ljcrf.edu> 
writes:
> has anybody tried to do blunt end ligation in the presence of a blunt-
> cutter? 
> 
> this should work for blunted DNA fragments that won't restore the 
> original site in the vector once ligated. The idea is to push the rxn 
> into the fragment ligated direction w/o having to CIP the vector. 
> 
> ->      which enzymes do work? 
> 
> ->      which buffers did you use? 


Helge, I try to avoid subcloning fragments that are blunt at BOTH ends.  
Otherwise you end up just recircularizing the vector without insert unless, 
of course, you phosphatase the vector.  If I was in a bind, I would rather 
put linkers on the blunt ends so that I could make them into sticky ends.

Inserting a fragment that is blunt on only one end and sticky on the other is 
very easy.  Any two blunt ends will go together just fine, but in most cases 
you will lose the ability to cut at that site with either enzyme.  But you 
may create a new restriction site in the process!  If you clone into a 
polylinker, it doesn't matter if you lose the site at that position because 
there is usually another unique site from the polylinker nearby.

Hope this helps.

‹ Brian