curious ligation question...

Robot-Boy s535290 at aix1.uottawa.ca
Thu Feb 5 19:35:58 EST 1998


hi all,

maybe this is a really dumb question, but let's say you are setting up a
ligation with vector that (in theory) shouldn't ligate to itself (ie.cut
with two different enzymes, needlessly dephosphorylated, *and* gel
purified). You set up two ligations with a test insert, use fixed
amounts of vector : one ligation has 5 times more insert than the other
one. I haven't mentioned anything about vector/insert ratios because for
my experiment, the amount of real insert is *very* limited. Anyway...these
test ligations were done so that I could get an idea as to the number of
transformants expected when the amount of insert is as limited as mine is.
Anyhow, barring any mislabelling of my tubes, I obtained a slightly higher
number of transformants from the ligation where the insert was was
"limiting". And this was surprising to me. I would have figured that 
the number of transformants obtained would increase linearly with
the amount of insert used, given the fact that in this case, the vector
was kept constant and it was in excess with respect to the insert.  Any
thoughts ?  Again, the only reason that I am using an excess of vector, is
the fact that my insert is limiting and I would think that the chances of
ligating a vector (that can't self-ligate) to a given insert would only
increase as you increase the amount of available insert for the ligation.

puzzled and bewildered....

TIA, 

Ed




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