Dna speedvac concentrator

Triplicate 10 mg samples were extracted using 400 μl of 80% (v/v) ethanol for 10 min in a BioShake at 100°C and 1,000 rpm. The extraction was performed three times and all ethanol soluble fractions were pooled after centrifugation. Total soluble sugar was determined using anthrone reagent (0.2% anthrone, in 70% H₂SO₄ v/v), and 1 mg/mL of glucose was used for the standard curve (Whan et al., 2014 (link)). Free glucose and fructose were measured according to the method described by Campbell et al. (1999) and sucrose was determined as described by Birnberg and Brenner (1984) (link). The soluble α-gluco-oligosaccharide was measured by taking 200 μl of the ethanol soluble fraction dried in a DNA SpeedVac Concentrator (DNA120-230, Thermo Fisher, Waltham, Massachusetts, United States) and then using the Total Starch Assay Kit (Megazyme) as described above.

DNA for all 0.5 months samples and a minority of 3 months samples were extracted just prior to qPCR processing. However, the majority of 0.5 months samples had DNA extracted concurrent with their sampling (up to 18 months previously) and were then frozen at −20°C until being thawed for this study. Following standard DNA extraction (Richardson et al., 2001 (link)), the DNA concentration of all samples was measured using a ThermoScientific NanoDrop8000 Spectrophotometer and sample concentration was to 20–30 ng/ml to ensure similar amplification of samples during qPCR. Where necessary, samples were diluted with T10E0.1 (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, pH 8.0) or concentrated using a ThermoScientific Savant DNA SpeedVac Concentrator. Purity of samples was checked through measurement of 260/280 absorbance ratios; ratios were between 1.7–2.0 for all samples.