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Team:Alberta/Methodology/Protocols
From 2011.igem.org
(Difference between revisions)
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<h2>Protocols</h2> | <h2>Protocols</h2> | ||
| - | <h3 id=extraction> | + | <h3 id=extraction>Extraction and Esterification</h3> |
<h4>Sample Preparation for GC Analysis:</h4> | <h4>Sample Preparation for GC Analysis:</h4> | ||
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motor and pestle </li> | motor and pestle </li> | ||
<li>add the <i>N. crassa</i> to a large screw top jar, and add methanol and methanolic HCL | <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. | + | <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>incubate samples in glass tubes in a 70 degree water bath for two hours</li> | ||
| - | <li>add | + | <li>add ½ the volume of the reaction of ddH20 </li> |
| - | <li>add | + | <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>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>repeat steps ix-x, and add to the rest of the hexane</li> | ||
Revision as of 03:34, 27 September 2011
METHODOLOGY
Protocols
Extraction and Esterification
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.
