Mobio powerlyzer power soil dna isolation kit

DNA extraction was performed using MoBio Powerlyzer Powersoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, USA). Samples that were immersed in solution were centrifuged at 13,000 × g for 5 min and the supernatant discarded. Samples were washed with 1 mL of cold 1× PBS (pH7.2) (Life Technologies, Melbourne, Australia) and centrifuged at 13,000 × g for 10 mins. DNA extraction was performed according to the manufacturer’s instructions with the following modifications. Samples were placed into bead tubes with solution C1 and heated at 65 °C for 10 min, prior to two cycles of bead beating at 6.5 m/s for 1 min using a FastPrep-24 bead beater (MP Biomedicals, Santa Ana, USA). Total DNA was eluted in 100 μL of sterile water. DNA concentration was quantified fluorometrically with a Qubit dsDNA HS Assay kit (Life Technologies).

Sediment core samples were collected from two adjacent wetlands, P7 and P8, at the United States Geological Survey-managed Cottonwood Lake Study Area near Jamestown, ND, USA [9 (link)]. From 16S rRNA gene analyses, 18 representative sediment samples were selected for metagenomic sequencing based on wetland (P7 and P8), season (winter, spring, summer), and depth (1–3, 10–12, and 19–21 cm) (Additional file 1: Table S1). After being stored at − 80 °C, sediments were thawed, and DNA was extracted using the MoBio PowerLyzer Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. Following extraction, nucleic acids were quantified (Additional file 1: Table S1) using a Qubit® Fluorometer (Invitrogen, Carlsbad, CA, USA) and diluted, so ~ 200 ng of DNA per sample was sent for metagenomic sequencing at the DOE Joint Genome Institute. These samples had been previously analyzed using 16S rRNA gene sequencing and pore water measurements of sulfate, sulfide, ferrous iron, methane, methanol, trimethylamine, ethanol, 2-propanol, acetate, acetone, and formate [9 (link)]. Here, these geochemical measurements were used as input values for principal component analysis in R [16 ] in order to illustrate the geochemical differences between P7 and P8.

Known masses of mouse feces were immersed in 1 ml of cold (4°C) 1× sterile phosphate-buffered saline (PBS; pH 7.4) (Thermo Fisher Scientific, United Kingdom) and centrifuged at 13,000 × g for 10 min to form a pellet for DNA extraction. The supernatant was transferred to a fresh microcentrifuge tube for metabolomics study. DNA extraction was performed using a Mo Bio PowerLyzer PowerSoil DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA, USA), as previously described (62 (link)).

Community DNA was extracted from faecal samples collected on the initial day of dosing (Day 0), and the day before the euthanization of the first animals (Day 9), as well as from ileal and caecal content using the MoBio PowerLyzer^® Power Soil^® DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA) according to the manufacturer’s recommendations with minor modifications: A maximum of 200 mg samples was used for extraction and samples were heated to 65°C for 10 min after addition of the C1 solution. Bead beating was conducted at 30 cycles/s for 4 min (Retsch MM 300 mixer mill). DNA concentrations were measured with the Qubit dsDNA HS kit (Life Technologies).

All laboratory protocols were performed at the Sackler Institute for Comparative Genomics at the AMNH. We performed DNA isolations and library preparations in a UV-sterilized laminar flow hood to minimize aerosol contamination. Intestinal tissue was scraped using sterilized razor blades. Guano and intestinal scrapings were placed in bead tubes and mechanically disrupted with a Disruptor Genie (Scientific Industries, Bohemia, NY) for 45 s⁻¹ min. We extracted microbial DNA from guano and intestinal mucosa samples using the MO BIO PowerLyzer™ PowerSoil® DNA Isolation kit (MO BIO Laboratories, Carlsbad, CA), using 0.25 mg of sample and following the manufacturer's instructions with the following amendment: samples were incubated at room temperature for two min on the extraction column membrane prior to final elution (QIAGEN, pers. comm.). Samples with high organic content were further purified using the PowerClean® Pro DNA Clean-Up Kit (MO BIO Laboratories, Carlsbad, CA). Each extracted DNA sample was quantified using a Qubit™ 2.0 Fluorometer and High Sensitivity dsDNA reagent kit (Invitrogen, Carlsbad, CA). A total of 55 DNA samples, 29 intestinal and 24 guano, were used for metagenomic library preparation, including extraction and PCR negative controls to account for contamination at each step in the library preparation.