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E. coli (Bl21DE3) lyses automatically

Albert Crescenzo atc at TRNA.CHEM.YALE.EDU
Tue Feb 18 13:08:28 EST 1997

	Our lab has had several problems with this.  The e.coli does not
like to express this particular protein.  Two things happen usually.  One,
the cells loose the plasmid (with amp selection) or they lyse.  try
switching to a vector that has kan instead of amp.  Also, try minmal
amounts of IPTG or don't induce at all (even the tightest repression is
leaky).  Slow the growth rate of the cells by growing in minimal media or
with very slow shaking (<100rpm).  Always use fresh transformants.  do not
store them on a plate.  keep the plasmid and trnsform for each expression
experiment.  hope this helps. 

Albert Crescenzo
Yale University
Dept. MB&B, Bass Rm 233
266 Whitney Ave.
New Haven CT, 06520
w-203 432-6206
h-203 937-7851

On Tue, 18 Feb 1997, Justinian wrote:

> On 17 Feb 1997, Huang Ke-xue wrote:
> > I met a serious problem recently. The transformed BL21(DE3)(by a expression
> > plasmid) lyses automatically after 6-8 hr growing. I repeated this three times. 
> > Do you think my stuff was contamined by Phage(?). 
> > Any suggestion?
> 	are you sure it's e.coli?

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