wrong orientation of ligated fragment

ChenHA hachen at bc.ic.ac.uk
Fri Jun 4 14:03:12 EST 1999

On Tue, 1 Jun 1999, Gregor Kopitar wrote:

> Hello!
> I am trying to ligate  1500bp fragment into 6200bp expression vector at Bgl
> ll restriction site. All of the positive clones have the fragment in wrong
> orientation and I do not have anymore ideas, what to do.
> I welcome any suggestion.

I don't know what expression system you are using, but if you are
using e.coli, a useful suggestion would be to use a T7 based
expression vector such as the pET series of vectors.  You problem
is most likely to be toxicity of the gene product to the cell, if you
do cloning in cell without T7 polymerase gene (that include all the
cells used for cloning I think), you won't have any problem with
toxicity.  Also choose a vector that gives you high level of
control of expression.  Now when it comes to expression you will have
a problem as it seems that low basal expression level is toxic.  One
possibility is to express the gene product by using phage to introduce
the T7 polymerase (Novagen do such a system I think) or the various
pLysS and pLysE cells. But you can worry about that after you've clone
the gene itself.

> Ursa Pecar, PhD Student
> ursa.pecar at ijs.si

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