Preferential insert orientation
Mon Jan 3 19:33:49 EST 1994
In article <gregoryh-271293155836 at 220.127.116.11> gregoryh at bcm.tmc.edu (Gregory R. Harriman) writes:
> 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
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 ?
Uni of Sydney
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