wrong orientation of ligated fragment

George Rutherford gruther at bilbo.bio.purdue.edu
Wed Jun 2 13:59:12 EST 1999


In article <199906011621.KAA16264 at nestor.NMSU.Edu>, hroychow at NMSU.EDU
("Hiranya S. Roychowdhury") wrote:


> 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....
> 
> 
> Dr. Hiranya Sankar Roychowdhury


If it's possible, he might also consider changing the host strain,
although I suppose it may be specific to the expression vector.


George



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