Ligation question, quite simple

David F. Spencer dspencer at is.dal.ca
Fri Nov 7 12:18:50 EST 1997


In article <3461802A.2A9 at nospam.ic.ac.uk>, k.desmet at nospam.ic.ac.uk wrote:

> 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
> > 
> > 

<< Explanation Snipped >>

> 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. 

NEB (New England Biolabs) uses precisely that criterion, i.e., after x-fold
overdigestion , x % of the DNA fragments can be ligated and recut, or
occasionally, after x-fold overdigestion, x % of the DNA fragments can be
ligated and x % of those recut.

Dave

--------------------------------------------
David F. Spencer, PhD
Dept. Of Biochemistry
Dalhousie University
Halifax, Nova Scotia
Canada

dspencer at is.dal.ca
dspencer at rsu.biochem.dal.ca



More information about the Methods mailing list