Another Ligation Problem

Richard J. Dudley rdudley at nrc.uab.edu
Wed Apr 24 12:48:22 EST 1996


It looks also like one of your enzymes isn't cutting very effectively.  
The buffers for the two enzymes from their respective companies are very 
different (I just looked).  This could lead to only one enzyme cutting, 
then the single cuts being recircularized.  The few without an insert 
could be the lucky few that have two cuts made in them, and there is too 
litle insert to favor oligomerization.  With this scnario, your chances 
of obtaining the desired clone are rather low.  I would suggest the 
following:
	1) try using a vector that has the polylinker in the opposite 
orientation, then directionally cloning.  I don't immediately see a 
vector that Promega has that is such, however.

	2) try cutting a large amount of the DNA, then purify the fragment 
(this will let you know whether or not both enzymes are cutting also).  
Then set up a ligation to favor oligomerization.

Good luck!

Richard J. Dudley
Neurobiology Research Center
University of Alabama School of Medicine





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