Restriction Enzyme Activity and Ligation Problems

Scott Fraser sfraser at raid.res.petermac.unimelb.edu.au
Wed May 21 19:51:41 EST 1997


	I've been having problems with my ligation of a 350 base pair PCR
fragment into a 7kb vector for quite some time now.  My PCR primers were
designed to incorporate the restriction enzyme site for Eag1 at both ends
of the fragment.  This enzyme provides an overhang which is compatible
with Not1 cut vectors, and is reported to give 100% efficiency of cutting
when the site is 2 base pairs of double stranded DNA from the end of
linear DNA. (Biotechniques Vol. 19, No. 1 (1995)).  I cut my fragment with
Eag1 (NEB) and ethanol precipitate, I then attempt to ligate into my Not1
cut vector (which has been CIP treated) with a 5:1 molar ratio of insert
to vector.  I incubate the ligations overnight at room temperature and
then cut with Not1 (as the Not1 site is destroyed with the insert is in
place) and then clean up and transfect.  I have been obtaining colonies on
my vector only but few or none on my cut vector controls and my test
ligations.  I believe the problem may be in the digestion of the PCR
fragment.  Therefore my question is two parts:
	Firstly has any one else there had problems with enzymes not digesting as
close to the ends as published?
	Secondly has anyone got a good method of telling if the enzymes have
digested, the difference is 6 base pairs? (I've tried high resolution gels
and would like to avoid acrylamide gels if possible)

	Also if any one has any advice on how I could improve my ligations all
suggestions would be appreciated.

Thanks in advance
Scott Fraser
sfraser at raid.res.petermac.unimelb.edu.au
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