fresh transformations for protein expression??

JG. Head bijgh at zeus.bris.ac.uk
Thu Jan 11 10:30:23 EST 1996


Michael Szardenings (msz at bio.embnet.se) wrote:
: 2. Many people are claiming, that these effects are due to plasmid
: instability. I have never seen any real proof for this, i.e. effects that
: go beyond the normal loss of plasmids due to selection for non-expressing
: clones. In the worst case I have seen growth arrest of the culture
: followed some time later by rapid growing cells without plasmid, as the
: ampicillin used in the experiment had been degraded during the initial
: growth.

I am expressing what I would expect to be non-toxic constructs using the 
pET system, and have had similar problems.  Freshly transformed cells 
express well but after a week siting in the fridge the cells won't 
express at all.  Even the glycerols I have made lost the ability to 
express protein.  Like Michael I'm not convinced that these problems are 
due to plasmid instability.  I've tried various experiments to test if 
the plasmid is unstable and have seen no evidence for it at all.  But if 
this loss of expression is not due to plasmid instability, what else 
could it be?

Any ideas?

Cheers,

Jared

--
Jared Head     at the Department of Biochemistry, University of Bristol



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