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IPTG concentration - (Feb/01/2006 )

Hi there,

I want to express my protein in BL21 cells but I was just wondering if using 3 microliter of IPTG 0,5mM is enough for 1 liter of culture.

Thanks.

-vik-

Typically IPTG induction is carried out at concentrations (final concentration in your LB) around 0.5-1mM.

hope it helps!

jsavas

-jsavas-

QUOTE (vik @ Feb 1 2006, 03:40 PM)
Hi there,

I want to express my protein in BL21 cells but I was just wondering if using 3 microliter of IPTG 0,5mM is enough for 1 liter of culture.

Thanks.


We┬┤re using for induction of the BL21 a stock solution between 200 and 400 mM IPTG, using 1 ml for 1 l culture results in a endconcentration of 0.2-0.4 mM.

-ms-olli-

Thanks to you two for your replies. smile.gif

Vik

-vik-

poeple who used same vector and ecoli (BL21) as I will use now used 0.2mM.....so ill stick to that and see what happens.... dry.gif

-Kathy-

Hi

Does anyone know what OD600 we should grow the cell to after adding IPTG? I read somewhere that it's suppose to reach saturation but Im not sure what this means.

Thanks

-dummypants-

hi

actually u should determine the optimal IPTG condition and how many hours after to keep for shaking and then the temperature at which u grow also affects ur protein expression


it differs for different proteins in BL21 , only u can determine that amount or temperature or how many hours



so first optimize it
try a range from 0.1--1mM IPTG at 37 for 4-5 hours after induction
if ur lucky to get the optimal IPTG concentration,

then use that concentration for a variation in time for which u incbuate after induction from 1- 7 hours

after that use that time and IPTG concentration to try at different temperatures like 20-25-30-37-40 etc

finaly u can say that ur protein is best expressed at what conditions

hope it helps

-phytoviridae-

generally IPTG induction is carried out for no more than 5 hours but it depends on your protein. sometimes, to reduce proteolysis/ degradation of the protein, you can induce at room temperature for about 3 hours, nice and easy. i've used both 3 hours at 25 degrees C, and 4 hours at 37 degrees C, the former purification was much cleaner in terms of degradation products; still, it's protein relative.

-pipet_overuse-