Meta g nome dna isolation kit

Five days after ligature placement, each ligature was recovered and vortexed in sterile PBS. Bacterial DNA was isolated using the Meta‐G‐Nome DNA Isolation Kit (Epicentre, Madison, WI, USA) and used for 16S rDNA analysis (BGI America, Cambridge, MA, USA). The V4 region of 16S rDNA was amplified by PCR for next‐generation sequencing library construction. Taxonomic classification was performed by Ribosomal Database Project Classifier v. 2.2.6

Slugs were collected from a suburban area in North Cheshire (53.391463 N, 2.211214 W), a sampling area used in a previous study (Joynson et al., 2014 (link)), 2 h after last light. Individuals were cooled to 4°C to reduce spontaneous mucus production during dissection. Whole gut tracts were extracted, and care was taken to avoid rupturing the gut wall, to minimize loss or contamination of gut juices. All dissections were carried out in a sterile petri dish. Ten gut tracts were then pooled and DNA extracted using a modified protocol based on the Meta-G-Nome DNA isolation kit (Epicentre, WI, USA). Briefly, gut pieces were homogenized in an extraction buffer by vortexing, and a series of centrifugation steps were then carried out to remove plant material from the gut and other large debris. Supernatants were then filtered through a 1.2 μm filter in order to capture eukaryotic cell debris followed by a microbe capture step using a 0.2 μm filter. Microbes were then washed off the filter and DNA was extracted. DNA quality and quantity was assessed spectrophotometrically (260:230 and 260:280 nm ratios) and using agarose gel electrophoresis alongside a pre-quantified fosmid control. Extracted DNA was then used to create an Illumina DNA library and sequenced using a Miseq using the V2 chemistry (2 × 250 bp) at the Centre for genomic research, Liverpool University.

Wild mosquito adults were collected by CDC mini light traps (BioQuip, USA) or artificial catching aspirator at livestock corrals from Jining and Caoxian County in Shandong Province, China in July, 2012. With the owners’ consent, the light traps were set up in cow pens from 18:30 pm to 8:30 am next day. Mosquitoes of the An. hyrcanus group were sorted out in the field by morphology using the identification keys [1 ], and kept individually in silica gel filled tubes at 4 °C until DNA extraction. After being brought back to the laboratory, the female adults were separated into head and body. The single head was used to identify species by PCR assay based on rDNA ITS2 sequences [5 (link)]. Twenty bodies pool of An. sinensis species was extracted genomic DNA using Meta-G-Nome™ DNA Isolation Kit (Epicentre, USA), followed by Illumina sequencing.

Total genomic DNA of oral biofilms was extracted using Meta-G-Nome™ DNA Isolation Kit (Epicentre Biotechnologies, Madison, WI, USA) according to the protocol of the manufacturer. Stool DNA was extracted using the QIAamp^® DNA Stool Mini Kit (Qiagen, Hilden, Germany). DNA quality and amount were determined using Qubit dsDNA HS Assay Qubit Fluorimeter 2.0 (ThermoFisher Scientific, Carlsbad, CA, USA).
The hypervariable V3–V4 region of 16SrRNA was amplified using the primers, Bakt_341F CCTACGGGNGGCWGCAG and Bakt_805R GACTACHVGGGTATCTAATCC [33 (link)]. Amplicons were sequenced by Macrogen (Seoul, Republic of Korea) by high-throughput sequencing using Illumina MiSeq 2 × 250 platforms (Illumina Inc., CA, USA). The sequence data are available at https://www.ncbi.nlm.nih.gov/bioproject/735261.