Team:Alberta/Methodology/Protocols

• Overview
• Parts
• Protocols
  • Growth
  • Genetics
  • Extraction and Esterification
• Notebook
  • Growth
  • Genetics
  • Esterification
• Safety
  • Section I.
  • Section II.
  • Section III.
  • Section IV.
  • References

Protocols

Extraction and Esterification

Sample Preparation for GC Analysis:

1. wash clean glass tubes with a 2:1 chloroform:methanol solution; drain solution and let evaporate in fume hood
2. set water bath to 70 degrees Celsius
3. weigh out day 5 N. crassa samples (~0.1-0.3g)
4. transfer samples to clean class tubes; add 2 mL of methanolic HCl to each sample
5. incubate samples in glass tubes in a 70 degree water bath for one hour
6. add 0.9% NaCl to each rxn tube and mix (rxn is stopped)
7. add 2 mL hexane_IS (0.5 mg/ml C15) to each glass tube, vortex 2 min @ max speed
8. spin @ 3000 rpm for 5 mins
9. 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:

1. wash clean glass tubes with a 2:1 chloroform:methanol solution; drain solution and let evaporate in fume hood
2. set water bath to 70 degrees Celsius
3. weigh out day 5 N. crassa samples (~10-30g)
4. dried out large mass of N. crassa and crushed with a motor and pestle
5. add the N. crassa to a large screw top jar, and add methanol and methanolic HCL
6. use 10x ml of liquid for mass of fungus. ¾ of liquid volume should is methanol, ¼ is 3M methanolic HCL.
7. incubate samples in glass tubes in a 70 degree water bath for two hours
8. add ½ the volume of the reaction of ddH20
9. add ½ the volume of hexane, and shake vigorously
10. Let settle and pipette off the top hexane layer, being careful not to take any of the bottom layer.
11. repeat steps ix-x, and add to the rest of the hexane
12. remove hexane under negative pressure.