Quant it dsdna br assay kit

For digesters R^TcSS and R³⁷SS, samples were withdrawn on days 9, 38, 80 and 99 of operation. The starting point for this experiment was set to the day when the digesters had been in operation for three HRTs at full load. For digesters R⁴⁴SS and R⁵²SS, samples were taken after operation at the corresponding temperature (44°C and 52°C, respectively) for around three HRTs, i.e. on days 224 and 402. For digester RM, samples were taken on days 42, 85 and 148. A 20 ml sample was withdrawn from each digester and stored at −20°C until analysis. Total genomic DNA was prepared in triplicate using the FastDNA Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA). Aliquots of 200 μl digester sludge were used for extraction following the manufacturer’s protocol, and 60 μl water was used in the final elution of DNA. The concentrations of extracted DNA were measured using the Quant-iT dsDNA BR Assay Kit (Invitrogen, Life Technologies Europe, Stockholm, Sweden).

Samples for nucleic acid extraction were collected from the reactors when methane was stably produced in the steady state of operation at applied HRT, i.e., after operation for 122 days (OLR: 5.9 g/L-reactor/day) and 200 days (OLR: 37.2 g/L-reactor/day) in reactor 1 and after operation for 159 days (OLR: 21.1 g/L-reactor/day) in reactor 2. Total DNA and RNA was extracted from the cell pellet collected from 42 mL of the fermentation liquid (for PF) and 1 cm² pieces of the CFT support media (for BF). DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA) according to the manufacturer’s instructions. DNA quality was assessed by agarose gel electrophoresis, spectrophotometric analysis, and the Quant-iT dsDNA BR assay kit (Invitrogen, Carlsbad, CA, USA). RNA was extracted using TRIzol reagent (Invitrogen) and was purified using an RNeasy Mini Kit and RNase-Free DNase Set (Qiagen, Valencia, CA, USA). The quality of extracted RNA was evaluated using an Agilent 2100 Bioanalyzer with RNA 6000 Pico reagents and RNA Pico Chips (Agilent Technologies, Santa Clara, CA, USA).

Samples were collected for 16S rRNA gene sequencing during the initial stable phase of the anaerobic digestion (after 68 days) and in the final phase (after 152 days) from reactors MDi, McoDi, TDi and TcoDi, in addition to the cow manure used for co-digestion. Food waste was also sampled for the same purpose, but genomic DNA was not successfully recovered, likely due to the sanitization treatment (70 °C for 1 hour). All samples were frozen immediately after sampling, and stored at −20 °C until DNA extraction. For DNA extraction, thawed samples were centrifuged at 18 800 x g for 7 min. to remove the liquid. The pellet was then re-suspended in S.T.A.R buffer (Roche Diagnostics Corporation, USA) to stabilize nucleic acid and prevent bacterial growth. The suspension was vortexed followed by a subsequently slow spin to dissociate cells from large particles. The cell-containing suspension was transferred to FastPrep24 tubes with acidic washed glass beads for mechanical lysis. DNA was extracted using a commercial DNA extraction kit (LGC Genomics, UK), and DNA concentration measured with Qubit™ fluorometer and Quant-iT™ dsDNA BR Assay Kit (Invitrogen, USA). The DNA samples were stored at −20 °C until sequencing preparation.

All the examined Stipa spp. specimens were collected during field research in the years 2011–2014 (see Supplementary Table S3). Total genomic DNA was extracted from desiccated leaf tissue using ZR Plant/Seed DNA MiniPrep^TM kit (Zymo Research Corp., USA) and Genomic Mini AX Plant Spin (A&A Biotechnology, Poland) following the manufacturers recommendations. DNA quality was assessed by the 1% agarose gel electrophoresis and quantity was estimated with the use of the Qubit fluorometer system and the Quant-IT ds-DNA BR Assay kit (Invitrogen, USA).4.

Plasmid DNA from Dub-seq library samples was extracted either individually using the Plasmid miniprep kit (Qiagen) or in 96-well format with a QIAprep 96 Turbo miniprep kit (Qiagen). Plasmid DNA was quantified with the Quant-iT dsDNA BR assay kit (Invitrogen). The BarSeq PCR of UP barcodes was done as previously described^{13 (link)} with ~50 ng of plasmid template per BarSeq PCR reaction. To quantify the reproducibility of both UP and DOWN barcodes in competitive growth experiments, we collected plasmid DNA from nickel and cobalt experiments, and amplified both UP and DOWN barcodes in two separate PCRs using the same plasmid library template. For BarSeq PCR of DOWN barcodes, we used universal-forward-primer DT_BarSeq_p1_FW and reverse primer DT_BarSeq_IT017. The PCR cycling conditions and purification steps were same as for the UP barcodes^{13 (link)}. All experiments done on the same day and sequenced on the same lane are considered as a “set”.