Preferential insert orientation

Mark Mark
Mon Jan 3 19:33:49 EST 1994


In article <gregoryh-271293155836 at 128.249.14.251> gregoryh at bcm.tmc.edu (Gregory R. Harriman) writes:
>Help!
>		I have been trying to reverse the orientation of a 3.2 kb insert which I
>have cloned into the Xba1 site of pKS.  To do this, the insert was cut out
>of pKS with Xba1, run on a low melt gel and purified.  It was then ligated
>with new pKS which has been digested with Xba1 and dephosphorylated.  All
>the appropriate controls for ligation, dephosphorylation, transformation,
>etc are okay.  Nonetheless, I only get colonies with the original
>orientation.  I have even digested the plasmid with Xba1 AND Xmn1 (the
>latter digests the pKS vector but not the insert) to be sure that no
>contaminating pKS is carried over with the insert and to ensure that small
>amounts of linearized plasmid do not remain in the insert prep.  Still no
>luck. Does anyone have any previous experience with this type of problem? 
>What can cause inserts with the same restriction site on both ends to
>ligate in only one direction? Any advice or helpful suggestions would be
>appreciated!

Hi I had a 2kb insert do the same. Well sort of
I found that when I was using blue/white selection that
one orientation was white and the other blue. I picked 
this up by doing a colony blot.

Is this a possiblity in your situation?
Can you do a forced cloning to give the orientation you want?
Have you thought about an adaptor ?
Mark Smith
Dept. Biochemistry
Uni of Sydney
Sydney, 2006
Australia



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