Double stranded dna hs assay kit

Genomic DNA from each isolate was isolated from overnight cultures using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA). The quality of the DNA was checked using a NanoDrop 1000 (Thermo Scientific, Rockford, IL) and the concentration was determined using a Qubit double-stranded DNA HS assay kit and a Qubit 2.0 fluorometer (Life Technologies, Grand Island, NY), according to each manufacturer's instructions.

The V4 region of the 16S rRNA gene was amplified, as described by Caporaso, Lauber [16 (link)]. Briefly, 16S rRNA amplification was performed in 25-μL reactions using 0.025 U Takara Hot Start ExTaq (Takara Mirus Bio Inc., Madison, WI), 1X Takara buffer with MgCl2, 0.4 pmol/μL of F515 and R806 primers, 0.56 mg/mL of bovine serum albumin (Roche Applied Science, Indianapolis, IN), 200 μM of dNTPs and 10 ng of genomic DNA. Reactions were performed in triplicate with the following: initial denaturation (98 °C, 2 min), 30 cycles of 98 °C (20 s), annealing at 50 °C (30 s), extension at 72 °C (45 s), and final extension at 72 °C (10 min). Amplicons were verified using a 2% Tris/Borate/EDTA agarose e-gel (Life Technologies, Grand Island, NY), cleaned and normalized using SequalPrep Normalization Plates (Applied Biosystems, Foster City, CA), and further quantified using the Qubit 2.0 Fluorometer and the double-stranded DNA HS Assay Kit (Life Technologies). Samples were pooled in equal moles at concentrations of 5 ng, purified using AMPure SPRI beads (Beckman Coulter, Brea, CA), denatured and diluted to 2 nM, and 5 pM was loaded onto the Illumina Nextseq cartridge with 40% (v/v) of denatured 12.5 pM PhiX spike-in control.