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MB Sample ID: SA093781
| Local Sample ID: | 6HKR5 |
| Subject ID: | SU001363 |
| Subject Type: | Plant |
| Subject Species: | Triticum durum |
| Taxonomy ID: | 4567 |
| Genotype Strain: | cv. Opera |
| Age Or Age Range: | 10 days |
| Gender: | Not applicable |
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Sample Preparation:
| Sampleprep ID: | SP001371 |
| Sampleprep Summary: | Freshly homogenized (100 mg) plant material were obtained for each biological sample (plant) and replicates. These were transferred to a 2 ml microcentrifuge round bottom screw cap tubes (Eppendorf). Extraction was done by adding 1400 µl of methanol (at -20°C) and vortexing for 10 s after addition of 60 µl ribitol (0.2 mg/ml stock in dH2O) as an internal quantitative standard for the polar phase. Samples were transferred in a thermomixer at 70C and were shaken for 10 min (950 rpm) and were then further centrifuged for 10 min at 11000 g. The supernatants were collected and transferred to glass vials where 750 µl CHCl3 (-20°C) and 1500 µl dH2O (4°C) were sequentially added. All the samples were vortexed for 10 s and then centrifuged for another 15 min at 2200 g. Upper polar phase ((150 µl ) for each replicate were collected, transferred to a 1.5 ml tube and were dried in a vacuum concentrator without heating. Before freezing and storing at -80°C, the tubes were filled with argon and placed in a plastic bag with silica beads (for avoiding moisture and hydration during short-term storage). Before derivatization, stored samples, were placed in a vacuum concentrator for 30 minutes to eliminate any trace of humidity. Then, to the dried samples, 40 µl methoxyamine hydrochloride (20 mg/ml in pyridine) were added and incubated for 2 h in a Thermomixer (950 rpm) at 37 C. Methoxyaminated samples were then silylated by adding 70 µl of MSTFA to the aliquots. Samples were further shaken for 30 min at 37 C. |
| Sampleprep Protocol ID: | NA |
| Sampleprep Protocol Filename: | NA |
| Sampleprep Protocol Comments: | NA |
| Processing Method: | Freshly homogenized (100 mg) plant material were obtained for each biological sample (plant) and replicates. These were transferred to a 2 ml microcentrifuge round bottom screw cap tubes (Eppendorf). Extraction was done by adding 1400 µl of methanol (at -20°C) and vortexing for 10 s after addition of 60 µl ribitol (0.2 mg/ml stock in ddH2O) as an internal quantitative standard for the polar phase. Samples were transferred in a thermomixer at 70C and were shaken for 10 min (950 rpm) and were then further centrifuged for 10 min at 11000 g. The supernatants were collected and transferred to glass vials where 750 µl CHCl3 (-20°C) and 1500 µl ddH2O (4°C) were sequentially added. All the samples were vortexed for 10 s and then centrifuged for another 15 min at 2200 g. Upper polar phase (150 µl ) for each replicate were collected, transferred to a 1.5 ml tube and were dried in a vacuum concentrator without heating. Before freezing and storing at -80°C, the tubes were filled with argon and placed in a plastic bag with silica beads (for avoiding moisture and hydration during short-term storage). |
| Processing Storage Conditions: | On ice |
| Extraction Method: | Freshly homogenized (100 mg) plant material were obtained for each biological sample (plant) and replicates. These were transferred to a 2 ml microcentrifuge round bottom screw cap tubes (Eppendorf). Extraction was done by adding 1400 µl of methanol (at -20°C) and vortexing for 10 s after addition of 60 µl ribitol (0.2 mg/ml stock in ddH2O) as an internal quantitative standard for the polar phase. Samples were transferred in a thermomixer at 70C and were shaken for 10 min (950 rpm) and were then further centrifuged for 10 min at 11000 g. The supernatants were collected and transferred to glass vials where 750 µl CHCl3 (-20°C) and 1500 µl ddH2O (4°C) were sequentially added. All the samples were vortexed for 10 s and then centrifuged for another 15 min at 2200 g. Upper polar phase (150 µl ) for each replicate were collected, transferred to a 1.5 ml tube and were dried in a vacuum concentrator without heating. Before freezing and storing at -80°C, the tubes were filled with argon and placed in a plastic bag with silica beads (for avoiding moisture and hydration during short-term storage). |
| Extract Enrichment: | NA |
| Extract Cleanup: | NA |
| Extract Storage: | 4℃ |
| Sample Resuspension: | Dried for Derivatization |
| Sample Derivatization: | Methoxyaminatin + trimethylsilylation (MSTFA + 1% TMCS) |
| Sample Spiking: | 60 µl ribitol (0.2 mg/ml stock in ddH2O) |
| Subcellular Location: | NA |
