eDNA Extraction from Water Samples

For decades, environmental microbiologists have developed, refined, and compared methods to capture and extract DNA from environmental samples [42] . We selected a widely used approach from aquatic microbiology wherein water samples are filtered through PCTE membranes and the filter retentate is extracted using a CTAB protocol wherein chloroform chemically dissolves the PCTE filter [43] (link), [44] (link). We chose PCTE membranes (47 mm diameter, GE Osmonics, sold by Barney Corporation, Hilliard, Ohio, USA) with a 10 µm pore size because in side-by-side trials one PCTE membrane allowed filtration of 2 L of water in approximately the same amount of time (∼10 min) required to filter 2 L of pond water through one 1.5 µm pore size glass fiber (GF) filter (47 mm diameter, grade 934-AH, Whatman, GE Healthcare Life Sciences, Piscataway, New Jersey, USA). The 934-AH GF filter and 2 L water volume are specified in the current standard operating procedure for eDNA-based monitoring of bigheaded carp [25] (link), [40] (link). Our CTAB DNA extraction protocol (Protocol S1) differs from that of Coyne et al. [43] (link), [44] (link) only in the absence of β-mercaptoethanol from the CTAB buffer, which we removed for convenience because it produces a strong disagreeable odor.