Shine Dalgano and T7 promoter

Dominic-Luc Webb molmed domweb at mbox.ki.se
Thu Aug 2 11:48:46 EST 2001


Thanks for your comments, I am reposting to the group
because your email address is giving errors.

Basically, we mutated in a true Shine Dalgano sequence,
AGGAGG. We sequenced and there is still a correct T7
promoter and the new Shine Dalgano, 8 bases upstream of
ATG. The insert is EGFP and I have transformed this into
our E coli, BL21(DE3) from Novagen.

Using a fluorescence spectrophotometer, 1 mM IPTG
treatment for 2-3 hours gave a small fluorescence
signal, characteristic of EGFP, so I think we got some
expression, finally.

I am curious, how much IPTG and what length of time is
typically optimal for protein expression?


Dominic




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