pulse-chase in E. coli
dkling at warren.med.harvard.edu
Mon Dec 20 10:58:30 EST 1993
Here is the response to the query:
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
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
if you would like - just email me.
Quite aside from my negative remarks above...I would be interested if
does get that sort of experiment to work. Could you post a summary to
Ohio State University, Department of Biochemistry
caroline+ at osu.edu
Thank you Caroline,
More information about the Methods