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I have several mg/mL of D-luciferin, luciferase (firefly), ATP solution (25 mmol) and MgSO4 (for the mg2+ ions). The experiment has to be done in cuvettes of 2,5 mL (if I get any results, it will be measured with a spectrophotometer).

I was wondering if you can give me an idea/tips/advice how I can recreate bioluminescence with those chemicals.

I am struggling with the ratio's of the chemicals, pH and whether I should dilute the solution or not.

I don't know how I should mix the ratio's of D-luciferin, luciferase, ATP solution etc. Next thing is pH, I dont know what the optimal pH will be of this mixture and how I can change the pH accurately. These two points go with dilution as well, because I want to use water as a solvent and for dilution, but I dont know if it is needed or not.

(Also, if you know how the other chemicals might impact the energy that will be absorbed in the mixture and how it will be released when you know that the emission wavelength of D-luciferin is around 530 nm in water, please share you knowledge if you want)

I would greatly appreciate it if someone can give me advice or lead me to the right direction on the problems that I am facing with this bioluminescence experiment. I have tried asking this to other people, but no one was willing to help me, please.

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