16S Amplicon Sequencing of Marsh Soil: Mesohaline Interior, Second Intermediate Depth
DNA was extracted from the collected soils (~0.60 g) using a Qiagen DNeasy PowerLyzer Powersoil Kit following the manufacturer protocol and the PowerLyzer 24 homogenizer option for bead beating (4000 rpm, 30 sec). Extracted DNA was quantified on a NanoDrop 2000 (Thermo Scientific) and diluted (nuclease-free H2O) to a final concentration and volume of 1.5 ng L-1 and 5 L, respectively. DNA was amplified by polymerase chain reaction (PCR) (Bio-Rad C1000 Touch ThermoCycler) using the 515FY (Parada et al. 2016) and 806R (Caporaso et al. 2011) primers targeting the 16S rRNA gene fragment. The PCR program ran at 95C for 3 min followed by 30 cycles at 95C for 30 sec, 55C for 1 min, and 72C for 1 min with a final step at 72C for 5 min. All PCR reactions consisted of 12.5 L of GoTaq MasterMix (Promega Corporation), 1 L of each primer (10 mM), and 5 L of DNA (1.5 ng L -1) with the rest of the 30 L solution comprised of nuclease-free H2O. Amplified genes were then sequenced using MiSeq Reagent Kits v2 (250 bp reads) on an Illumina MiSeq platform at the VIMS Genomic Facility.
SUB15044979
TC3_I2