wrong orientation of ligated fragment

Yuji Tai ytai at kms.ac.jp
Tue Jun 1 22:24:54 EST 1999


I am wondering if a mammalian expression vector in which a toxic
fragment inserted still affect the bacteria viablity. Does the CMV
promoter work in bacteria?

> I presume that you have confirmed this by sequencing.
> Anyway, there is nothing much one can do under the circumstances (single
> site ligation, I mean). What is concluded normally is that the esxpressed
> polypeptide in the correct (required) orientation is cytotoxic....
> Suggestion:
> clone the fragment in a bare-bone vector that does not have any leaky
> promoter and has a BglII site along with others. Cut it out of this clone
> with BglII and another RE (X). Clone it directionally into your expression
> vector at BglII/X sites.
> However, if the expressed prtoduct is indeed toxic to the cells, you are no
> further along....

Yuji Tai
ytai at kms.ac.jp
Kagawa Medical Univeristy
http://www.kms.ac.jp/~physiol1/staff/ytai



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