Reorganizing clones

Thomas Duchaine duchaint at vif.com
Thu Oct 23 00:55:34 EST 1997


Hi there!

	I've been cloning for a very long time amd i never observed as much
reorganized clones as i did with the C-terminal part of my protein. This
cloning have been tried from the product of a Vent pol PCR, digested on
one side by Hnd III (so producing a half sticky insert) and i tried to
use pMAL-C tm and pQE vectors (quiagen). Trying different vectors and
bacterial strains, i still observe aberrant patterns of migration and
still have been unable to clone this (/$%"%??"?*!!!) sequence. This part
of my protein is supposed to interact with other proteins but under the
cloning conditions, its not supposed to be expressed. Even using a
plasmid producing an efficient repressor for the promotor. So it does
not look like i do a leaky expression. I also tried bacterial strains
that are supposed to be deficient for the recombination machinery, same
thing...

		I'm desperate!! Anybody knows about what i could have done to
advantage such reorganization and more importantly, how i can get rid of
it?? PLEASE!

				Thomas.



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