Blunt end ligations - another question

Rafa Maldonado Rafael at genetics.med.utah.edu
Wed Feb 9 20:30:11 EST 1994


Sorry, Brian, but you are wrong.
It is possible cloning with two blunt ends if you adjust the conditions
of the ligation (amount of DNA, insert/vector ratio and units of
enzyme, and considering the relative size of the insert and the
vector), more or less as Maniatis says. I remember some old IBI catalog
that gave perfect protocols for every case of ligation, and it worked
fine. Where did those protocols go? IBI forgot them many time ago.
Other buffers from different companies have PEG that enhances ligation,
especially blunt-end (BRL, I think). Indeed, it is possible advoid the
CIP for almost every ligation, using the right conditions. I only use
CIP for very special cases. i.e. exchanging identical fragments from a
plasmid to another, cloning very big inserts in very small vectors, and
so.
The Helge's question is different, and I don't know if it'll work. But
it sounds great; it woul be used in every ligation that implies
disapearing sites, not only blunt-end cutters, but in all compatible
enzymes. However, I don't find the relation of cloning in this way with
the CIP; have I missed something? 
Other different question: is it possible dephosphorilate the vector for
a blunt cloning, and stil having enough yield of ligation? I thought
that in blunt-end ligations dephosphorilation gives very (VERY) low
eficiencies.

Bye

Rafa
----------------------------------
Rafael Maldonado
Howard Hughes Medical Insitute
University of Utah
Rafael at genetics.med.utah.edu



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