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