Team:Caltech/Notebook/material/Biofuel Protocols

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     <h2><a name="Biofuel_Protocols" style="color: #000000";>Biofuel Protocols</a></h2>
     <h2><a name="Biofuel_Protocols" style="color: #000000";>Biofuel Protocols</a></h2>
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<h3><a name="Ethanol_Assay";>Ethanol Assay</a></h3>
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<h3><a name="Ethanol_Assay"; style="color: #000000";>Ethanol Assay</a></h3>
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<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>
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<h5>Ethanol Color Assay Reaction</h4>
<img src="https://static.igem.org/mediawiki/2012/1/11/Ethanol_assay_reaction.png" alt="Photo of Reaction" title="Ethanol Color Assay" height="200px" width="700px" />
<img src="https://static.igem.org/mediawiki/2012/1/11/Ethanol_assay_reaction.png" alt="Photo of Reaction" title="Ethanol Color Assay" height="200px" width="700px" />
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<h4>Materials</h4>
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<ul>
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<li> water
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<li> 1.0M bicine (pH 8.0)
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<li> 40 mM EDTA
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<li> 10 mM NAD
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<li> 4.2 mM thiazolyl blue
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<li> 16.6 mM phenazine ethosulfate
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<li> 1 mg/mL alcohol dehydrogenase enzyme
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<li> ethanol
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</ul>
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<h4>Method</h4>
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<p> Make a master mix consisting of:</p>
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<ul>
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<li> 3.6 mL water
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<li> 1.2 mL bicine
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<li> 1.2 mL EDTA
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<li> 1.2 mL NAD
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<li> 1.2 ml thiazolyl blue
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<li> 2.4 mL phenazine ethosulfate
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</ul>
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<p> Before running the experiment, add 1.2 mL of alcohol dehydrogenase enzyme. </p>
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<p> Pipette 5 uL of sample into the 96-well microtiter plate.  Add 95 uL of master mix. 
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Measure absorbance at 570 nm.  We found that the maximum absorbance of each well occurred
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at a different time relative to the concentration, so it is best to run a series of measurements
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over a period of approximately 4 hours. </p>
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<br>
     <a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/material#Materials_and_Methods";>Back to Materials and Methods</a>
     <a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/material#Materials_and_Methods";>Back to Materials and Methods</a>
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<h3><a name="MOPS"; style="color: #000000";>MOPS Buffer</a></h3>
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<p>
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We used the recipe from Teknova listed <a href="http://www.teknova.com/EZ-RICH-DEFINED-MEDIUM-KIT-p/m2105.htm"> here </a> but substituted 10 ml of 20% glucose for 8 ml of 50% glucose.  Mix all the following ingredients together to get MOPS buffer:
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</p>
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<ul>
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<li> 100 ml 10X MOPS Mixture </li>
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<li> 10 ml 0.132 M K2HP04 </li>
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<li> 100 ml 10X ACGU </li>
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<li> 200 ml 5X Supplement EZ </li>
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<li> 582 ml Sterile H20* </li>
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<li> 8 ml 20% Glucose </li>
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</ul>
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    <a style="float:right;" href="https://2012.igem.org/Team:Caltech/Notebook/material#Materials_and_Methods";>Back to Materials and Methods</a>
 +
 +
</body>
</body>

Latest revision as of 23:16, 17 July 2012



Biofuel Protocols

Ethanol Assay

Ethanol Color Assay Reaction
Photo of 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.


Back to Materials and Methods

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
Back to Materials and Methods

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