Ligation question, quite simple

Dr. Duncan Clark duncan at genesys.demon.co.uk
Fri Nov 7 12:23:52 EST 1997


In article <3461802A.2A9 at nospam.ic.ac.uk>, Koen De Smet
<k.desmet at nospam.ic.ac.uk> writes
>It is a way of stating how pure the restriction enzyme is. With one 
>unit, you should get 100% digestion of 1 ug reference DNA. This should 
>produce nice sticky ends and these should nicely ligate back to each 
>other again (>95%). 
>
>If you used 25 times too much restriction enzyme, then you would also 
>increase any contaminating enzymes such as DNAses, polymerases etc. 
>These may affect the sticky ends, and the DNA may then fail to ligate. 
>But if you still get 95% religation, it suggests that there is very 
>little/no contamination with such other enzymes.
>
>It is not foolproof however. If sticky end get converted to blunt ends, 
>they will still ligate. A better test would be to check if the ligated 
>DNA can then be redigested with the enzyme. This would confirm that the 
>sticky ends were not affected and the RE site recreated. 

Conversely, just because an RE won't liogate don't assume that it has
contaminants. Some blunt cutters ie Nru I and single base overhang
cutters ie BstN I ligate really poorly. This is just a characteristic of
the enzyme itself.

All RE's are checked for cut/ligate/recut.

Duncan

-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288
http://www.dnamp.com
http://www.genesys.demon.co.uk



More information about the Methods mailing list