pET15b (Novagen) IPTG induction

Paul J. Phelan via methods%40net.bio.net (by Paul.Phelan from tufts.edu)
Thu Sep 4 22:25:47 EST 2008


I am having trouble with inducing expression of a protein that I have 
cloned into pET15b (Novagen).  I have tried induction in BL21 E. coli 
with 1.0 mM IPTG in LB/100 ug/ml Ampicillin, at an OD(600 nm) of 
0.5-0.6, at 37 C for up to 8 hrs and then overnight.  Harvested cells 
are lysed by 30 min. incubation with 1 mg/ml lysozyme, followed by 
sonication (6x 10s for small scale 4 ml lysates, prepared from up to 
100 ml cultures).  But so far, my IPTG-induced lysates on SDS-PAGE look 
just the same as a non-induced control; I see no IPTG-dependent band at 
52 kDa where I want to see one.  I have re-sequenced my plasmid after 
transformation into BL21, and the sequence is correct.  Am I missing 
something somewhere?

Any enlightenment would be greatly appreciated

Paul Phelan
Tufts University
Department of Biochemistry
Boston



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