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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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MOPS Buffer

We used the recipe from Teknova listed here but substituted 10 ml of 20% glucose for 8 ml of 50% glucose. Mix all the following ingredients together to get MOPS buffer:

• 100 ml 10X MOPS Mixture
• 10 ml 0.132 M K2HP04
• 100 ml 10X ACGU
• 200 ml 5X Supplement EZ
• 582 ml Sterile H20*
• 8 ml 20% Glucose

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