1. Methanol Extraction:

Extraction Mixture
20:2:1 MeOH:water:ribotol solution
Ribitol 0.2 mg/ml in water

Metabolite Extraction Protocol

  1. Labelle 2 mL ependorf tubes with MeOH resistant marker and add ball bearing.
  2. Tare scales on tube with ball, harvest and weigh tissue (~100 in tube and rapidly freeze in liquid nitrogen.
  3. Homogenise tissue in ball mill for 2 min at 16. Place back into liquid nitrogen and homogenize for a further 2 min.
  4. Add 500 μl of extraction mixture (precooled to -20) and incubate at 70 C for 15 min at 1200 rpm on the thermomixer.
  5. Centrifuge at full speed for 3 min
  6. Take 400 µl of supernatant into new tube and discard pellet
  7. Dry down 60 µl in a new tube in the speedivac (~1hr), store remainder under argon at -80.
Derivitisation
  1. Add 20 µl of 20 mg/ml methoxyamine hydrochloride in pyridine (made fresh in fumehood) and incubate at 30 C for 90 min 1200 rpm on the thermomixer in fumehood.
  2. Add 30 µl MSTFA and incubate at 37 C for 30 min at 1200 rpm on the thermomixer in fumehood
  3. Add 10 µl of retention index markers (stored in SMS -80)
  4. Transfer entire volume to glass vials ready for GCMS analysis.
2. Fatty acid analysis:
  1. Collect plant tissue (50 to 100 mg) into a 2 ml round bottom Eppendorf tube containing grinding ball, snap-freeze using liquid nitrogen and grind into a fine powder using ball mill pre-cooled by liquid nitrogen (See protocol for plant tissue collection and processing).
  2. Add 400 μl of isopropyl alcohol with 0.01% BHT (2,6-di-tert-butyl-4-methylphenol, as antioxidant) and 50 μg of C19:0 internal standard to the tube and heat at 75oC for 15 min (to inactivate enzymes).
  3. After cooling down to room temperature add 600 μl of n-Hexane to the tube and sonicate for 5 min.
  4. Centrifuge the extract for 5 min at maximum speed and take 500 μl of supernatant into a fresh microcentrifuge tube.
  5. Open lid and dry supernatant to completion in SpeedVac at RT (~3hr).
  6. To the residue add 300 μl of Methanol with 2.0 % of H2SO4 (v/v) and heat at 80oC for 2hours.
  7. After cooling down to room temperature add 300 μl of 0.9% NaCl to the mixture followed by 300 μl of Hexane. Vortex, then centrifuge at top speed for 30s. Carefully transfer the hexane layer to a GC/MS vial for subsequent analysis.

logos The Government of Western Australia The University of Western Australia Australian Research Council Centre of Excellence in Plant Energy Biology


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