Cohesive End Ligations - Question

WUSC wusc at bscr.uga.edu
Wed Jul 3 09:44:00 EST 1996


In article <Pine.A32.3.91.960702103135.81003A-100000 at aruba.ccit.arizona.edu>, Kathy L <kathryn at U.Arizona.EDU> writes:
>
>Is it possible to run cohesive end ligations too long - i.e. would you 
>end up with high-molecular weight concatomers?  I'm getting some 
>high-weight stuff when I run a gel and the transformations aren't working.
>
It's possible, but it usually does not matter because the ligation results
depend on the total ligatable ends in your ligation mix. If you're doing
simple cloning in plasmid, chances are you'll always get the right
recombinant DNA (circular DNA with your insert ligated to the vector) IF
THE LIGATION WORKS.  If you're dealing with lambda vectors, the larger
concatomer, the better.
I did encounter once the same problem, though, with STRATAGENE T4 ligase.
I solved the problem simply by throwing the Stratagene into the trash and
using enzymes from other sources (e.g. Biolabs).
Hope this is helpful, but further communication is welcome.

Sheng-Cheng
wusc at bscr.uga.edu
 



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