electroporation problem

Normand normand.jolicoeur at nrc.ca
Thu Apr 20 13:47:07 EST 2000


Hi !

We are currently constructing a random oligonucleotides library. We have
to ligate the oligos to a plasmid containing a particular gene and then
use the contructions to transform DH5 bacteria. The constructions are ok
as determined on gel. We need to use a transformation protocol with high
efficiency, because we want the library to be as good as possible. So,
we decided to use electroporation. But, we encounter a strange problem.
When we transform the bacteria with the parental plasmid, our
transformation efficiency is very good. But, when we transform with the
random oligos containing plasmid, we cannot get any transformants. We
tried to mix the parental plasmid with the oligos (without ligation),
just to see if our oligos purification method generates some toxic
residues. But, everything is ok. When we transform the oligos containing
plasmid by heat-shock, it's also normal. It seems that, when the oligos
are ligated to the plasmids, they somehow inhibit the transformation by
electroporation. Does someone as any tips about this strange phenomena ?

Thank you.

Normand Jolicoeur

Biotechnology Reserach Institute, Montreal

email: normand.jolicoeur at nrc.ca





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