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Biofuel Protocols

Ethanol Assay

Ethanol Color Assay Reaction

Materials

• water
• 1.0M bicine (pH 8.0)
• 40 mM EDTA
• 10 mM NAD
• 4.2 mM thiazolyl blue
• 16.6 mM phenazine ethosulfate
• 1 mg/mL alcohol dehydrogenase enzyme
• ethanol

Method

Make a master mix consisting of:

• 3.6 mL water
• 1.2 mL bicine
• 1.2 mL EDTA
• 1.2 mL NAD
• 1.2 ml thiazolyl blue
• 2.4 mL phenazine ethosulfate

Before running the experiment, add 1.2 mL of alcohol dehydrogenase enzyme.

Pipette 5 uL of sample into the 96-well microtiter plate. Add 95 uL of master mix. Measure absorbance at 570 nm. We found that the maximum absorbance of each well occurred at a different time relative to the concentration, so it is best to run a series of measurements over a period of approximately 4 hours.

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