Preferential insert orientation

dkatz at earth.doane.edu dkatz at earth.doane.edu
Tue Dec 28 14:50:45 EST 1993


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.. 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!

If this is the vector I'm thinking it is - Stratagene's pBluescriptKS, why not
use the other direction vector - the SK - to clone into .  You would have
reversed the orientation, and be able to differentiate the original from the
new, since the primers would be reversed?
Or am I missing the point (my cataloges are downstairs), and there is no
other way to do it?
Sue Katz, DKatz at Doane.edu



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