Sybr green rt pcr master mix

Species-specific primers were designed (Table S6) and tested against other species in the community. Templates for qPCR analysis were prepared as described below. In brief, 1 ml of (co-)culture was centrifuged, and pelleted cells flash-frozen and stored until analysis. Frozen samples were re-suspended in 1 ml of deionized water, 400 μl of mixture transferred into a polypropylene screw cap tube containing 0.5 ml of acid washed glass beads (0.2-0.3 mm). Homogenization was done in a FastPrep-24 bead beater (4 m/s setting, for 2 min with intermittent cooling on ice). Lysate was diluted 1000 times with an alkaline PEG reagent (60% PEG 200, 20 mM potassium hydroxide, pH 13.3-13.5 as described in (Chomczynski and Rymaszewski, 2006 (link)) and incubated for 10 min at 95°C. 1 μl of resulting sample was used directly for qPCR reaction.
qPCR reaction mix was prepared using 1 μl template and 19 μl of SYBR Green RT-PCR master mix (Life Technologies), 0.5 μM primers (synthesized and desalted by Sigma-Aldrich). Amplification was performed on StepOne Plus real-time PCR system (Applied Biosystems), 40 cycles, 60°C annealing temperature. Quantification was done against serial dilutions of corresponding monocultures of a known optical density, with a standard curve generated on every microtiter plate. Data analysis was done using StepOne software (Applied Biosystems).

Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Germany). RNA purity and concentration were estimated with an ND-1000 spectrophotometer (NanoDrop Technologies, Thermo Scientific, UK). Total messenger RNA was converted to cDNA using the Promega Reverse Transcription System (A3500, Promega) as described by the manufacturer. Validated primers used (Sigma-Aldrich UK) are listed in Supplemental Table 1 & 2. qRT-PCR was carried out using SYBR® green RT-PCR master mix (Life technologies) according to the manufacturer's guidelines. PCR reactions with 50ng/μl of the cDNA samples per 10μl final reaction volume, were performed using standard cycling parameters (Stage 1: 50°C for 2min, Stage 2: 95°C for 10min then 40 cycles of 95°C for 15 Sec and 60°C for 1 min) on an ABI 7900HT sequence detection system. GAPDH was used as endogenous control and the DMSO solvent control sample used as the calibrator for each independent repeat. Data analysis using the ΔΔCt Method was carried out using SDS 2.2 software (Applied Biosystems).