Ligation question, quite simple

Koen De Smet k.desmet at nospam.ic.ac.uk
Thu Nov 6 03:30:34 EST 1997


Wolfgang Kemmner wrote:
> 
> Hi all,
> 
> we are doing (first time!) some ligation reactions. Now the manual says:
> after 25-fold overdigestion, 95% of the DNA fragments can be ligated.
> What do they mean: 25 fold more restriction enzyme, or what ?
> 
> Thanks for simple answers
> Wolf
> 
> 
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. 


Koen De Smet
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