Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.

pET15b (Novagen) IPTG induction

pET15b (Novagen) IPTG induction - Protocols and Methods Forum

pET15b (Novagen) IPTG induction - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.

LinkBack Thread Tools Display Modes
Old 09-05-2008, 03:25 AM
Paul J. Phelan
Posts: n/a
Default pET15b (Novagen) IPTG induction

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

Reply With Quote
Old 09-09-2008, 05:04 PM
Posts: n/a
Default pET15b (Novagen) IPTG induction

In article <[Only registered users see links. ].net> , "Paul J. Phelan" <[Only registered users see links. ]> wrote:

If the above is really correct, you won't see induction. BL21 cells
don't have T7 DNA polymerase required to read off T7 promoter
found in pET plasmids. You need to use BL21(DE3) strain that
has replication-deficient T7 phage in its chromosome. Or any other
(DE3) strain.

If you did use DE3, there are several other options like protein
found in inclusion bodies (give milky appearance after cell
lysis) or protein being cytotoxic and cells expressing it being
selected against during overnight culture due to the slightly leaky
promoter. In which case the overnight must be done in rich defined
medium containing glucose (MDGA, for example; part of the
autoinduction protocol).


Reply With Quote
Old 09-17-2008, 08:55 PM
Posts: n/a
Default pET15b (Novagen) IPTG induction

On Sep 4, 8:25*pm, "Paul J. Phelan" <[Only registered users see links. ]> wrote:

Hi Paul,

You want to use BL21(DE3) cells and not just BL21. The former cells
contain a prophage carrying the T7 RNA polymerase gene and lacIq.
When you add IPTG to the culture, it binds to the lac repressor coded
by the lacIq, allowing expression of the T7 RNA ploymerase which in
turn binds to the T7 promoter on the pET plasmid and turns on genes
downstream of the promoter.

Good Luck!
Reply With Quote

induction , iptg , novagen , pet15b

Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Problems with IPTG induction of PT7/lac Operon Pipetti13 Recombinant Protein Forum 0 07-29-2009 07:10 PM
IPTG induction aftabac Microbiology Forum 5 03-05-2009 02:47 AM
pET15b (Novagen) IPTG induction E. Lis Protocols and Methods Forum 0 09-08-2008 11:00 AM
pET15b (Novagen) IPTG induction Ho-Leung Ng Protocols and Methods Forum 0 09-05-2008 06:34 PM
IPTG induction in non E.coli strains Francesca Protocols and Methods Forum 22 03-24-2007 12:17 AM

All times are GMT. The time now is 02:59 AM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2015, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12211 seconds with 16 queries