q

Michael Benedik bchs1b at Elroy.UH.EDU
Wed Feb 3 14:39:28 EST 1993


In article <1993Feb2.134647.1 at agri.huji.ac.il>, navot1 at agri.huji.ac.il writes:
>I am trying to clone a dimer of the TYLCV virus whose coat protein was
>replaced with the lux ab gene encoding for luciferase.
>I have the construct in the form of a monomer in Puc 118, and the
>lux ab was active as tested in DH5 alpha cells using decanol and exposing
>trhe bacteria an autoradiogram.  My bacterial stock of this clone has always
>been uniformely active till a few days ago when I began to see a large
>variation in level of the luciferase activity between the colonies.
>Has anyone had similar experiences, or explain to me what is going on.
>My other problem is that I am trying to clone the construct as a head to tail
>dimer in the  pBin vector.  I have managed to clone the monomer but not a dimer
>The construct is 4000bp long and the pBin is apporoximately 10 kb so that a
>dimer in the pBin woul be 18 kb.  Does any one know if there is a limit to the
>size insert the pBin can accept?
>Any advice would be appreciated.
>Ilana
>

Most plasmids don't really have a size limit when propagated in E. coli,
however the larger they get the less efficiently they transform. but
cosmids are just pBR322 with a cos site, so 45-50kb plasmids certainly
work. (But they can be a pain to get good DNA preps from sometimes.)
As to  why your colonies are showing variation, that is hard to explain
without knowing more details. Most likely your cultures may be losing the
plasmid in some population of cells. Make sure your ampicillin is fresh
and your are using fresh plates. An alternative is that you are getting
mutants which no longer express luciferase. You might want to retransform
from a known good stock of DNA onto fresh amp plates. Oftentimes if you
are picking colonies which have been sitting around on plates for a while
you can get lots of variants.

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 Michael Benedik				INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik at uhou
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