Preferential insert orientation

Gregory R. Harriman gregoryh at bcm.tmc.edu
Mon Dec 27 17:18:04 EST 1993


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!



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