Deletion dilemma

WILLIS at BCRSSU.AGR.CA WILLIS at BCRSSU.AGR.CA
Tue Feb 6 16:24:24 EST 1996


Dear Netters:

	I've recently been attempting to produce deletions in a 
large (19kb) plasmid with very little success. Clones I isolate
are typically very small (approx. 3kb). The original construct 
is stable, although I encountered similar problems (but milder)
when I originally constructed it. The insert is a 16kb mouse 
mt genome in the vector pSP64 (amp resistance) with a T7 promotor
cassette. 
	To produce deletions I digest with a single enzyme
(I've tried several), gel purify the band (typically 10-16 kb
including the vector), perform a room temp. ligation and transform
chemically competent dh5alpha. Plating results in a very large
number of tranformants but all without the full length insert. I 
observe a variety of different colony morphologies when I plate. 
Previously I used this to my advantage, as the full length construct
formed a very small colony. Unfortunately (with the deletions)
this is no longer the case. I've selected dozens of colonies to 
date and they are all severely deleted. 
	I have tried changing cell strains (JM109 and Sure (which
are recB recJ) but this resulted in _no_ transformants.
	I would appreciate any suggestions. Thanks in advance.

Les Willis
Agriculture Canada
Summerland, B.C.
V0H 1Z0
Willis at BCRSSU.AGR.CA






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