Qubit dsdna hs high sensitivity assay

Filters from all water and sediment samples were extracted using Qiagen PowerWater© kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions with one exception: the final DNA elution step, was performed twice using 50 μL of DNA elution buffer (EB) for a final extraction volume of 100 μL. For stream transport, both 0.45 μm filters were initially processed separately during the lysis steps but loaded into the same spin column resulting in a single DNA extract per sample. DNA concentration for all samples was measured by fluorometric quantification (Qubit® High Sensitivity dsDNA HS Assay, Thermo Fisher Scientific, Waltham, MA), and DNA quality was measured using NanoPhotometer 260/280 ratio (NanoPhotometer Pearl, Implen Inc., Westlake Village, California). DNA extracts were stored at -80°C. Extraction blanks were performed 3 times throughout stream transport and mesocosm extractions and 1 time for nearshore samples; all extraction blanks were free of round goby DNA.

Water samples (1000 or 2000 mL) for eDNA studies were filtered through 1.5-μm glass microfiber filters (GE Healthcare Life Sciences, Pittsburgh, PA) following Nathan et al. (2015) and mesocosm tank water samples (50 mL) were filtered through 0.22-μm nitrocellulose filters (EMD Millipore, Billerica, MA). Filters were placed in Mo Bio PowerWater® bead tubes (MO BIO Laboratories, Inc., Carlsbad, CA) and held at -20°C until processing. DNA was extracted directly from the filters using Mo Bio PowerWater® kit according to manufacturer’s instructions with one exception: the final DNA elution step included two sequential rinses of the column with 50 μL each of PW6 for a final volume of 100 μL. DNA concentration for all samples was measured by fluorometric quantification (Qubit® High Sensitivity dsDNA HS Assay, Thermo Fisher Scientific, Waltham, MA), and DNA quality was measured using Nanophotometer 260/280 ratio (Nanophotometer Pearl, Implen Inc, Westlake Village, CA).