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MB Sample ID: SA035031
Local Sample ID: | 461_C_3 |
Subject ID: | SU000645 |
Subject Type: | Photosynthetic organism |
Subject Species: | Chlamydomonas reinhardtii |
Taxonomy ID: | 3055 |
Genotype Strain: | Wild Type |
Species Group: | Microorganism |
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Sample Preparation:
Sampleprep ID: | SP000652 |
Sampleprep Summary: | Harvested cells were flash frozen in liquid N and freeze dried. Accurately measured 50 ± 0.5 mg of freeze dried powder was used for metabolite extraction. Sample powder was pulverized with a single steel ball using TissueLyser LT (Qiagen) at 50 Hz speed for 5 min. One milliliter of extraction buffer containing MeOH:CHCl3:H2O (5:2:2; v/v/v; pre-cooled at -20 °C) was added and vortexed for 5 min. Ribitol (0.2 mg/mL in water; 10 µL) was spiked in the extraction buffer as internal standard in order to identify potential chromatographic errors. The homogenized material was centrifuged at 14000 rpm for 5 min and the supernatant was transferred to new tubes. 400 µL of pure water was added to the supernatant, samples were vortexed and centrifuged at 14,000 rpm for 5 min. The upper polar phase was transferred to new tubes for GC-MS analysis. An aliquot of 300 µL was dried out in vacuum concentrator without heating. To the dried material, 10 µL methoxyamine HCL in 100% pyridine (40 mg/mL) was added and shaken at 30 °C for 90 minutes and subsequently 90 µL of MSTFA 1% TMCS was added for trimethylsilylation of acidic protons and shaken at 37 °C for 30 minutes. The reaction mixture was transferred to GCvials with glass microinserts and closed by crimp caps. GC-MS data acquisition was performed as per previously published report (Wase et al., 2014) |