Embarssing problem

[kaj.stenberg from helsinki.fi.invalid]

Maurizio Affer <borderline86 from yahoo.com> wrote:
> Hi there
> I'm trying to express a small peptide in E. coli.
> After doing my cloning in Dh5alpha using a pgex-6p1
> vector (amersham) driven by a TAc prmoter I moved the
> plasmid into a Bl21-De3 strain.
> The problem is I discovered that I'm not even able to
> induce expression of GST (which is in the vector of
> course)!
> I induce at an OD of 0.8 with IPTG final of 0.1mM for
> 2 hours.
> All the trouble shooting are about "not enough protein
> induced", but here I'm at "no GST induction"!
> Please help whatever comes to your mind let me know...

Have you tested different media/induction time, they can vary
surprisingly much?

For one project (using BL21-De3) I did some testing with the following
results: 
	Induction time (0.1mM IPTG at OD 0.8): 
10% after 2h
90% after 15h, best at 20 h

Using the long induction time, media/temperature:
LB 37 degrees <5%
LB 28 degrees 50%
TB 37 degrees 100%
TB 28 degrees 70%
YT 37 degrees 10%
YT 28 degrees 25% 

So although the protocol you use is the standard one the optimal
parameters can vary a lot!


-- 
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Kaj Stenberg, Ph. D
Department of Biosciences            tel. +358-9-191 59682
Division of Biochemistry             fax +358-9-191 59068
P. O. Box 56, Viikinkaari 5          e-mail: kaj.stenberg from helsinki.fi
FIN-00014 University of Helsinki
Finland

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