DNA Extraction
Upon return to the laboratory, the preservative solution was removed from the sterivex filters and any DNA containing material captured on the filter membrane was recovered by addition of 720 µL of ATL lysis buffer and 40 µl proteinase K to the filter, followed by incubation at 56°C in a water bath with regular mixing by vortexing. The supernatant was then extracted using the DNeasy blood and tissue kit (Qiagen) following the manufacturer’s instructions with final resuspension in 200µl of elution buffer. All DNA samples were quantified using a Qubit 3.0 Fluorometer (Invitrogen) following the manufacturer’s instructions then stored at -20 ⁰C before use.
Each snail specimen was individually transferred to a clean, sterile mortar and ground into a fine paste using a pestle and liquid nitrogen. For some snail species, the individual specimens were pooled prior to grinding (see Table 1). After use mortar and pestles were immediately immersed in 10 % bleach for a minimum of 10 minutes and then cleaned in between samples with 10 % Distel (Tristel™), rinsed with dH2O and then autoclaved at 121 ⁰C for 15-20 minutes. DNA was extracted by the DNeasy blood and tissue kit (Qiagen) -with a final elution volume of 50 or 200µl - in a separate laboratory remote from water sample extraction and qPCR set up, using dedicated tissue extraction equipment. Disposable laboratory coats were worn, and benches and equipment were wiped down with a 10% bleach solution before and after use. Extracted DNA was quantified using a Qubit 3.0 Fluorometer (Invitrogen) following the manufacturer’s instructions then stored at -20 °C before use.