title: Sample 1
organism: mixed sample
description: Protocols: The river soil samples were collected in 50 mL sterile conical tubes, in triplicate, at a distance of 3-5 meters from the banks of the Potomac River and a 6-inch depth from the soil surface. Ethanol and paper towels were used interchangeably between each soil sample collection to ensure clean, sterile tools. Samples were transported to the laboratory within an hour of collection and stored in a -20°C refrigerator until further processed. The tubes were ultrasonicated for 1 minute each to achieve cell disruption. Extraction was completed using a Qiagen DNeasy Power Soil kit protocol. Approximately 0.25g of each soil sample was weighed and recorded. To achieve cell disruption, the samples were ultrasonicated for 1 minute each. Samples were then stored at -20°C until PCR was performed. Nanodrop was used to confirm the quality and concentration of the DNA obtained from the soil samples. DNA products were PCR amplified using primers “515F–806R” targeting the V4 region of the 16S SSU rRNA - used by the Earth Microbiome Project. Primer sequences are as follows: 515F (Parada)–806R (Apprill), F: GTGYCAGCMGCCGCGGTAA; R: GGACTACNVGGGTWTCTAAT. Primers and primer constructs were designed by Greg Caporaso. Modifications to primer degeneracy were done by the labs of Jed Furhman and Amy Apprill. Forward-barcoded constructs were redesigned by Walters. Amplification conditions were performed in a 25 µl reaction volume and consisted of 13 µL nuclease-free water, 10 µL of 2x PCR master mix, 0.5 µL forward primer, 0.5 µL reverse primer, 1 µL soil DNA. The thermal cycle was programmed for 120 seconds at 94°C as initial denaturation, followed by 35 cycles of 45 sec at 94°C for denaturation, 60 sec at 50 °C as annealing, 90 sec at 72 °C for extension, and a final extension at 72 °C for 10 min.