blunt ended ligation

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Wed Dec 28 11:36:07 EST 1994


Shayan Sharif writes:

> I have been trying to do a blunt-ended ligation, but it doesn't seem to 
> be working. ...  Moreover, agarose gel 
> analysis, using supercoiled marker, shows that the DNA in ligation 
> mixture is still in linear form.
> ... [did lots of good controls]

Look into the restriction reaction that generated the blunt ends in
the first place.  Typically the enzymes are contaminated with
exonucleases that will destroy the ligatability of the ends.  The
manufacturers purify these away to within limits specified in the spec
sheet, typically such that you can overdigest 100x and still get 90%
ligation.  However, it's easy to get beyond these limits.  For
example, if you put 1 ul (say 20 U) in with 0.2 ug vector and digest 3
hrs., you've just overdigested 300 x and it may not be ligatable. 
Also, some restriction enzymes just come with lousy specs to start
with.  And, of course, if the DNA is hard to cut because its not pure
in the first place, you're in a double bind, and the impurities in the
DNA may also include exonuclease.  Besides checking the spec sheet and
cutting the vector (and insert) with no more than 10-20 x excess if
possible, I recommend trying some other blunt end enzymes and maybe
some other DNA (like lambda) just to establish a positive control for
your ligation conditions using your gel assay.

Also, make sure you removed or killed the restriction enzyme before
the ligation step.  Some enzymes survive heat treatments; this is
usually spelled out in the manufacturer's catalogue.

Good luck,

Steve Hardies, Dept. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu




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