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.