Merge branch 'ceplas-dm-viktoria' into 'main'

Ceplas dm viktoria

See merge request !7

assays/CMML_18-0008_GC-MS/protocols/GC-MS.md

+## Metabolite profiling
+
+Approximately 50 mg of ground leaf material was used to obtain extracts for metabolite profiling by gas chromatography-mass spectrometry (GC-MS) as described by Fiehn et al. (2000). Data analysis was performed using MassHunter Software, version B.07.00 (Agilent). For relative quantification, all metabolite peak areas were normalized to the peak area of the internal standard ribitol added prior to extraction and the amount of leaf material. From each sample, two extracts were prepared and analysed.
\ No newline at end of file

assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md

+## Protein and Starch Measurements
+
+Approximately 25 mg of ground leaf material was used for extraction in 80% (v/v) ethanol with 10 mM 2-(N-morpholino)ethanesulfonic acid (pH 6). Extraction was performed three times with 500 µl extraction buffer while shaking for 1 hour at 90 °C. After each extraction step, samples were centrifuged at 12,000 g for 10 min and the supernatant was collected and pooled with previous fractions. From each sample, two extracts were prepared and stored at –20 °C until analysed. The protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
+
+Pellets obtained during protein extraction were gelatinized with 300 µl 0.2 M NaOH at 90 °C for 40 minutes. After adjustment to pH 5–6, 200 µl hydrolysis buffer was added [20 mM acetate buffer, pH 4.8; 0.5 U α-amylase (10102814001, Roche), and 4 U amyloglucosidase (A7095, Roche)] and the mixture was incubated overnight at 37 °C. Glucose resulting from starch degradation was measured enzymatically: 10 µl of digest was added to 100 mM HEPES-NaOH (pH 7.5) with 10 mM MgCl2, 1 mM NADP+, 2 mM ATP, 0.1 U glucose-6-phosphate dehydrogenase (G6PDHII-RO, Roche), and 4 U hexokinase (11426362001, Roche). The final reaction volume was 200 µl and the increase of absorbance at 340 nm was monitored.
\ No newline at end of file

assays/TitratableAcidity/protocols/TitratableAcidityProtocol.md

+## Titratable acidity
+
+Approximately 30 mg of ground leaf material was incubated in 500 µl 50% (v/v) methanol at 90 °C while shaking. After centrifugation at 13,000 g for 5 min at room temperature, 450 µl of the supernatant was collected and the remaining pellet was extracted once more. Both fractions were pooled and stored at –20 °C until use. Titratable acidity was measured using bromothymol blue (Carl Roth) as a pH indicator: 60 µl of extract was mixed with 40 µl distilled H2O and 4 µl bromothymol blue (1 mg/ml stock). Using the microinjector of a Synergy H1 (BioTek) plate reader, samples were titrated with 1 mM KOH in 5 µl steps. After each step, absorbance at 445 nm and 615 nm was recorded, and the ratio of the absorbances at 615/445 nm was calculated. Based on the 615/445 ratio of buffer standards (pH 4.6–7.5), the pH of analysed extracts was determined. From the volume of KOH required to titrate extracts to pH 7.0, the titratable acidity was calculated. Two extractions were performed from each sample, and each extract was titrated in three replicates.
\ No newline at end of file