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appropriate time for induction (adding IPTG)


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#1 Puteri Q

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Posted 13 August 2010 - 12:01 AM

Hii Bioforumers...

I need your help to deal with my research problem. It is about recombinant gene expression.

Based on the paper, induction of genes in E. coli (BL21) growing in Luria Bertani usually performed after reaching OD600 between 0.5-0,6. It is said to be coincide with the exponensial phase.
If we use another growth medium for expression, then we induce the culture with IPTG, should we know what time the bacteria reaching exponensial phase? or is it enough for us to add IPTG into the culture just when OD600 between 0.5-0.6?

Please help me to overcome this problem.

-_-

#2 protolder

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Posted 29 August 2010 - 09:43 PM

Hola, it depends of the composition of medium, the concentration of inducer etc. Sometimes the addition of inducer stops the growth. Well, in LB the max OD. reaches about 3 OD. units. You have to know how efficient is your medium to grow. For instance, TB reaches about 6-8 OD units. If I were you I would seed an ON culture withouth induce and in the morning I would mesure two diferent times to see if the OD has get stationary phase; supose that is 6-8 units, so you can induce at 1.5 or 2 OD units. Two more considerations if you seed a more rich media, you have to buffering it because the changes of pH could stop the growth. And if you assay your medium ON, take a sample to see if your protein is expresed withouth induction. The current BL21 isnīt LacIq so the expression isnīt hardly repressed. Good luck




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