pET, lysE, lysS, bacterial expression questions

Don Jackson djackson at welchlink.welch.jhu.edu
Thu Jun 9 10:57:30 EST 1994


In article <1994Jun6.141347.65336 at kuhub.cc.ukans.edu>,
peterg at rnaworld.bio.ukans.edu (Peter Gegenheimer) wrote:

> In <ma.42.000E7520 at ABC.univie.ac.at>, ma at ABC.univie.ac.at (Manuel Simon) writes:
> >In article <Cqs4LF.4y5 at ucdavis.edu> jfh <jfhess at ucdavis.edu> writes:
> >>From: jfh <jfhess at ucdavis.edu>
> >>Subject: pET, lysE, lysS, bacterial expression questions
> >>Date: Thu, 2 Jun 1994 17:00:51 GMT
> >
I've had very good results using the pET11 vector in BL21(DE3) plysS cells
(straight BL21's probably would have worked but I was too lazy to prep
some).
We use N-Z amine broth with 1x M9 salts and 0.4 % glucose (I think this is
described in Studier's Analytical Biochem. paper).  This plasmid has a lac
operator controlling expression of the insert.  It's otherwise identical to
pET3.  Novagen (who sells it) does recommend higher IPTG concentrations (1
mM vs. 0.4) and longer induction times (I usually use about 3 hours).  I
also make a practice of using freshly transformed colonies to innoculate my
cultures; I find this works better than frozen stocks.

Don Jackson
Johns Hopkins School of Medicine
djackson at welchlink.welch.jhu.edu 
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