bassuk at u.washington.edu
Thu Mar 26 00:46:30 EST 1998
yup. i suspect your MOI is poor (multiplicity of infection). although
reserved for viral or phage work, what this means is that your glycerol
stock is not 100% recombinant. try growing your bug in 2x antibiotics
in LB, then streak out with antibiotics, then repeat the liquid and
plate procedure 5 times! make a bunch of gycerol stocks. put some in
-20. put some in -80. use only the -20 for a month or so.
this is the only way to get reproducible results.
i assume you have paid attention to OD, temp, salts, pH, amount of
O2, amoutn of CO2, ete etc
On 11 Mar 1998, Henri M.H. Spronk wrote:
> We are trying to express a eukaryotic cDNA in a prokaryotic expression
> system. The E. coli strain is M15 [pREP4] from QIAgen. The pQE40 expression
> vector consist of 6His-mouseDHFR and our cDNA (220 bp). Between the
> mouseDHFR and the cDNA we placed a factor Xa-digestion site. In the
> beginning we had poor expression. At one moment, however, we obtained very
> good expression. We always used the same glycerolstock which is defreezed
> before used. So repeated freezing and defreezing of the bacteria seems to
> result in better expression. Does anybody understand this?
> After changing the Xa-site (4 aa) into AspPro (2 aa) expression is again
> very low. We tried already lower expression temperature and different
> incubation times. Repeated freezing and defreezing didn't result in a
> better expression. Can someone help us?
> Thanks in advance,
> Henri M.H. Spronk
> Department of Biochemistry
> University Maastricht
> henri.spronk at bioch.unimaas.nl
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