wrong orientation of ligated fragment

Hiranya S. Roychowdhury hroychow at NMSU.EDU
Tue Jun 1 11:27:24 EST 1999


At 03:56 PM 6/1/99 +0200, 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.
>
>
>Ursa Pecar, PhD Student
>ursa.pecar at ijs.si

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
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




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