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| | <h2>Esterification and Extraction</h2> | | <h2>Esterification and Extraction</h2> |
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| - | <h3>Procedures</h3>
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| - | <h4>Sample Preparation for GC Analysis:</h4>
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| - | <ol style="list-style-type:lower-roman">
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| - | <li>wash clean glass tubes with a 2:1 chloroform:methanol
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| - | solution; drain solution and let evaporate in fume
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| - | hood</li>
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| - | <li>set water bath to 70 degrees Celsius</li>
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| - | <li>weigh out day 5 <i>N. crassa</i> samples (~0.1-0.3g) </li>
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| - | <li>transfer samples to clean class tubes; add 2 mL of
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| - | methanolic HCl to each sample </li>
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| - | <li>incubate samples in glass tubes in a 70 degree
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| - | water bath for one hour </li>
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| - | <li>add 0.9% NaCl to each rxn tube and mix (rxn is
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| - | stopped)</li>
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| - | <li>add 2 mL hexane_IS (0.5 mg/ml C15) to each glass
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| - | tube, vortex 2 min @ max speed </li>
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| - | <li>spin @ 3000 rpm for 5 mins</li>
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| - | <li>carefully take the tubes out and transfer the top phase (hexane_IS) into a fresh glass tube; <b>IMPORTANT: do not disturb the intermediate layer</b></li>
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| - | </ol>
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| - | <p>(Hexane_IS NU-CHEK Prep Inc. LOT# A-615-J30-)</p>
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| - | <h4>Production of raw fuel:</h4>
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| - | <ol style="list-style-type:lower-roman">
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| - | <li>wash clean glass tubes with a 2:1 chloroform:methanol
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| - | solution; drain solution and let evaporate in fume hood</li>
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| - | <li>set water bath to 70 degrees Celsius</li>
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| - | <li>weigh out day 5 <i>N. crassa</i> samples (~10-30g)</li>
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| - | <li>dried out large mass of <i>N. crassa</i> and crushed with a
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| - | motor and pestle </li>
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| - | <li>add the <i>N. crassa</i> to a large screw top jar, and add methanol and methanolic HCL
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| - | <li>use 10x ml of liquid for mass of fungus. ¾ of liquid volume should is methanol, ¼ is 3M methanolic HCL.</li>
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| - | <li>incubate samples in glass tubes in a 70 degree water bath for two hours</li>
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| - | <li>add ½ the volume of the reaction of ddH20 </li>
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| - | <li>add ½ the volume of hexane, and shake vigorously</li>
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| - | <li>Let settle and pipette off the top hexane layer, being careful not to take any of the bottom layer. </li>
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| - | <li>repeat steps ix-x, and add to the rest of the hexane</li>
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| - | <li>remove hexane under negative pressure. </li>
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| - | </ol>
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| | </div> | | </div> |
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PROJECT
Esterification and Extraction
"
