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Team:Alberta/EsterificationExtraction
From 2011.igem.org
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<h2>Esterification and Extraction</h2> | <h2>Esterification and Extraction</h2> | ||
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| + | <h3>Procedures</h3> | ||
| + | |||
| + | <h4>Sample Preparation for GC Analysis:</h4> | ||
| + | <ol style="list-style-type:lower-roman"> | ||
| + | <li>wash clean glass tubes with a 2:1 chloroform:methanol | ||
| + | solution; drain solution and let evaporate in fume | ||
| + | hood</li> | ||
| + | <li>set water bath to 70 degrees Celsius</li> | ||
| + | <li>weigh out day 5 <i>N. crassa</i> samples (~0.1-0.3g) </li> | ||
| + | <li>transfer samples to clean class tubes; add 2 mL of | ||
| + | methanolic HCl to each sample </li> | ||
| + | <li>incubate samples in glass tubes in a 70 degree | ||
| + | water bath for one hour </li> | ||
| + | <li>add 0.9% NaCl to each rxn tube and mix (rxn is | ||
| + | stopped)</li> | ||
| + | <li>add 2 mL hexane_IS (0.5 mg/ml C15) to each glass | ||
| + | tube, vortex 2 min @ max speed </li> | ||
| + | <li>spin @ 3000 rpm for 5 mins</li> | ||
| + | <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> | ||
| + | </ol> | ||
| + | |||
| + | <p>(Hexane_IS NU-CHEK Prep Inc. LOT# A-615-J30-)</p> | ||
| + | |||
| + | <h4>Production of raw fuel:</h4> | ||
| + | |||
| + | <ol style="list-style-type:lower-roman"> | ||
| + | <li>wash clean glass tubes with a 2:1 chloroform:methanol | ||
| + | solution; drain solution and let evaporate in fume hood</li> | ||
| + | <li>set water bath to 70 degrees Celsius</li> | ||
| + | <li>weigh out day 5 <i>N. crassa</i> samples (~10-30g)</li> | ||
| + | <li>dried out large mass of <i>N. crassa</i> and crushed with a | ||
| + | motor and pestle </li> | ||
| + | <li>add the <i>N. crassa</i> to a large screw top jar, and add methanol and methanolic HCL | ||
| + | <li>use 10x ml of liquid for mass of fungus. ¾ of liquid volume should is methanol, ¼ is 3M methanolic HCL.</li> | ||
| + | <li>incubate samples in glass tubes in a 70 degree water bath for two hours</li> | ||
| + | <li>add ½ the volume of the reaction of ddH20 </li> | ||
| + | <li>add ½ the volume of hexane, and shake vigorously</li> | ||
| + | <li>Let settle and pipette off the top hexane layer, being careful not to take any of the bottom layer. </li> | ||
| + | <li>repeat steps ix-x, and add to the rest of the hexane</li> | ||
| + | <li>remove hexane under negative pressure. </li> | ||
| + | </ol> | ||
| + | </div> | ||
</div> | </div> | ||
Revision as of 03:10, 27 September 2011
PROJECT
Esterification and Extraction
Procedures
Sample Preparation for GC Analysis:
- wash clean glass tubes with a 2:1 chloroform:methanol solution; drain solution and let evaporate in fume hood
- set water bath to 70 degrees Celsius
- weigh out day 5 N. crassa samples (~0.1-0.3g)
- transfer samples to clean class tubes; add 2 mL of methanolic HCl to each sample
- incubate samples in glass tubes in a 70 degree water bath for one hour
- add 0.9% NaCl to each rxn tube and mix (rxn is stopped)
- add 2 mL hexane_IS (0.5 mg/ml C15) to each glass tube, vortex 2 min @ max speed
- spin @ 3000 rpm for 5 mins
- carefully take the tubes out and transfer the top phase (hexane_IS) into a fresh glass tube; IMPORTANT: do not disturb the intermediate layer
(Hexane_IS NU-CHEK Prep Inc. LOT# A-615-J30-)
Production of raw fuel:
- wash clean glass tubes with a 2:1 chloroform:methanol solution; drain solution and let evaporate in fume hood
- set water bath to 70 degrees Celsius
- weigh out day 5 N. crassa samples (~10-30g)
- dried out large mass of N. crassa and crushed with a motor and pestle
- add the N. crassa to a large screw top jar, and add methanol and methanolic HCL
- use 10x ml of liquid for mass of fungus. ¾ of liquid volume should is methanol, ¼ is 3M methanolic HCL.
- incubate samples in glass tubes in a 70 degree water bath for two hours
- add ½ the volume of the reaction of ddH20
- add ½ the volume of hexane, and shake vigorously
- Let settle and pipette off the top hexane layer, being careful not to take any of the bottom layer.
- repeat steps ix-x, and add to the rest of the hexane
- remove hexane under negative pressure.
