pulse-chase in E. coli

David Kling dkling at warren.med.harvard.edu
Mon Dec 20 10:58:30 EST 1993


Here is the response to the query:

Hello netters,

I would like to know whether anybody has had any experience in doing
pulse-chase experiments in E. coli using the T7 polymerase overexpression
system described by S. Tabor and F.W. Studier.  I have had trouble chasing
out the label after a 1hr incubation at 37 degrees.  I would appreciate any
useful information that you may have.  Please send responses directly to my
E. mail address.  I will post all the responses together.  

My E. mail address is dkling at warren.med.harvard.edu.

Thank you in advance!



Just a very quick thought about the pulse-chase experiments you propose - 
overexpressed T7 RNA polymerase is severely toxic *in the presence of the 
corresponding promoter*.  i.e. the polymerase itself is OK, but you put in
the 
polymerase plus a T7 promoter and the cell's rNTPs are rapidly depleted.

I read this in one of the papers from Tabor's lab.  I can look up the
reference
if you would like - just email me.

Quite aside from my negative remarks above...I would be interested if
anyone 
does get that sort of experiment to work.  Could you post a summary to 
methds-reagnts, please?

Regards,
Caroline Breitenberger
Ohio State University, Department of Biochemistry
caroline+ at osu.edu

Thank you Caroline,

d kling



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