Sample preparation workflow for NucleoSpin eDNA Water. Samples can be obtained either by filtration with round filters or filter cartridges (e.g., Sterivex). DNA is liberated from the filter by a strong lysis buffer. Alternatively, eDNA can be precipitated from <40 ml of unfiltered water (using Buffer PREC, Cat. # 740568). NucleoTrap Suspension beads efficiently bind the DNA while minimizing carryover of contaminants. Following washing and drying steps, the bead suspension is transferred to a NucleoSpin XS column, from which pure eDNA is eluted. The purification steps take less than 70 min, while total preparation times vary depending on the sampling and/or filtration setup.

Consistent performance with diverse round filters. 500-ml samples of creek water were filtered through the following different types of round filters, which were then processed using NucleoSpin eDNA Water: regenerated cellulose (RC), cellulose nitrate (NC), cellulose acetate (CA), polyester (PE), polycarbonate (PC), mixed cellulose ester (CM), and paper. As demonstrated by the resulting Ct values obtained in a downstream qPCR analysis, performance of DNA isolation was similar for each of the filter types and comparable to the performance observed with the recommended ethylene oxide-treated glass fiber filter (GF), which provides the best flow rate and lowest risk of clogging.

Compatibility with different water filtration systems. Samples of creek water were filtered through a glass fiber filter (GF) as well as several filtration systems commonly used in conjunction with eDNA purification. The NucleoSpin eDNA Water kit was then used to purify eDNA from each filter unit, and eluates were analyzed by qPCR for metazoan DNA. Purification from each filtration system yielded comparable results, as demonstrated by the consistent Ct values.

Superior downstream performance as compared to competitor kits. eDNA was purified from running water (creek) and static water (pond) using the NucleoSpin eDNA Water kit in parallel with two competitor kits (“BT” and “PW”) and analyzed by qPCR for metazoan DNA. As demonstrated by the lower Ct values, NucleoSpin eDNA Water provided higher sensitivity than the competitors for both sample types.

Seasonal traceability of European toad in a freshwater pond. In Germany, European toads become active after hibernation—usually in March—and typically start spawning in April. The presence of toads and/or spawn and tadpoles can be monitored by isolation of eDNA and subsequent PCR analysis. The NucleoSpin eDNA Water kit was used in combination with NucleoSpin Filter Midi to isolate DNA from a freshwater pond at the indicated timepoints, and PCR was performed to detect toad-specific or general eukaryotic DNA using corresponding primer pairs.