Shine Dalgano and T7 promoter

Kevin Brady kevin at basil.chem.nott.ac.uk
Fri Aug 3 04:18:30 EST 2001


You may find it better to optimise your expression by doing a matrix
experiment. It gives you a busy day, but the results are worth it:

Set up a bunch of cultures from your stock, and induce them under a
series of varied conditions e.g. something I did recently -

Set up 10ml cultures, all induced when OD600 = 0.6 (You could vary this
too if you wanted). Have a non-induced control too.
Varied conc. of IPTG 0.1mM 0.4mM 1mM 2mM
Varied temperature of induction 25 degs, 30degs and 37degs.
Did a time expression profile by removing 1ml samples every hour (0hrs,
1hr, 2hr, 3hr, 4hr, 5hr & o/n) and prepared SDS-PAGE samples from each.
Then gel the lot of them, gave me a really good profile for expression
conditions (which in my case gave two possibles 0.4mM IPTG @ 37degs for
3hours, or 1mM IPTG @ 25degs overnight)

There is no hard and fast rule for all protein expression systems, so
you really should optimise it yourself, believe me, one day of a
nightmare experiment makes the rest of your life easy :)

Cheers
Kev

--
Dr Kevin Brady
Organic Chemistry Section
School of Chemistry
University of Nottingham
Nottingham
UK
NG7 2RD
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Email : Kevin.Brady at nottingham.ac.uk or
        kevin at basil.chem.nott.ac.uk
URL   : http://www.nottingham.ac.uk/~pcxsc/thomas
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