curious ligation question...
Koen De Smet
k.desmet at nospam.ic.ac.uk
Fri Feb 6 03:44:20 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....
IMHO, if you have a vast excess of vector, then you increase the chance that your
insert will get a different molecule of vector at either end. So you will get a
linear piece of DNA: vector-insert-vector. Since you dephosphorylated your vector,
this will not be able to circularise. This will not be able to replicate in E.coli.
Do you have any idea of the amount of insert you have? YOu may have more success if
you use an equimolar ratio of insert:vector. Since you have only a small amount of
insert, you will need a small amlount of vector. To get colonies, you will probably
need supercompetent (commercial) E.coli.
Koen De Smet
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Imperial College School of Medicine at St Mary's
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