Shine Dalgano and T7 promoter

Luis Antonio Alcaraz Mas Luis.Alcaraz at ua.es
Thu Aug 2 13:35:23 EST 2001



Dominic-Luc Webb molmed escribió:

> 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

0.4 mM IPTG and 3 hours.

Luis.






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