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	<h4>Materials</h4>		<h4>Materials</h4>
	<ul>		<ul>
-	<li> 100 uL ethanol	+	<li> water
-	<li> 5 mL water	+	<li> 1.0M bicine (pH 8.0)
		+	<li> 40 mM EDTA
		+	<li> 10 mM NAD
		+	<li> 4.2 mM thazolyl blue
		+	<li> 16.6 mM phenazine ethosulfate
		+	<li> 1 mg/mL alcohol dehydrogenase enzyme
		+	<li> ethanol
	</ul>		</ul>
		+
	<h4>Method</h4>		<h4>Method</h4>
-	<p>The ethanol assay uses the following reagants and consists of the reaction depicted below. MTT Formazan appears purple, and it is due to this color change that the assay works.</p>	+	<p> Make a master mix consisting of:</p>
	<ul>		<ul>
-	<li>Make a serial dilution of ethanol. As shown below, we found a linear concentration to absorbance relation for concentrations under .05% ethanol.	+	<li> 3.6 mL water
-		+	<li> 1.2 mL bicine
		+	<li> 1.2 mL EDTA
		+	<li> 1.2 mL NAD
		+	<li> 1.2 ml thiazolyl blue
		+	<li> 2.4 mL phenazine ethosulfate
	</ul>		</ul>
-		+	<p> Before running the experiment, add 1.2 mL of alcohol dehydrogenase enzyme. </p>
		+	<p> 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. </p>
	<br>		<br>

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Revision as of 18:32, 3 July 2012

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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 thazolyl 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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