Coastal Environmental DNA Sampling & Gravity Filtration Protocol

Clean 1000 mL Nalgene bottles (as many as field blanks needed) with a 10% bleach rinse (leave for 10 minutes), then three rinses of DI water

Fill cleaned Nalgene bottles with 1000 mL of MilliQ water to use as field blanks

Sterilize luer lock to hose barb adapters, male-female luer lock caps, and silicone rubber cap

Fill a container (e.g. old pipette tip boxes, so you can easily drain the water with the lid) with 10% bleach solution, add adapters and caps, and let sit for 10 minutes

Rinse adapters and caps 3 times with DI water

Place adapters and caps in a single layer in UV Crosslinker for 15 minutes

Store sterilized caps and adapters in sterile containers

Wipe down plastic storage bins with RNase Away, and organize needed field supplies (wiping individual items with RNase Away when possible as well).

• Sampling Infrastructure & Transport: clothes rack, bricks for stability, shade structure, beach cart
• Tools: tube cutter, flathead screwdriver, lab marker, temperature and salinity probe (e.g. Orion Model 1230 meter), prepared field blanks
• Sampling Materials (1/sample) : zip ties, 1000 mL Covidien single-use enteral feeding pouches, sterile 0.22 μm Sterivex filters, male luer lock to hose barb adapters, dual male-female luer lock caps, round silicone rubber caps, sterile 3 mL luer lock syringes, small bungees, whirl-pak bags
• Preservation Materials: insulated cooler, ice packs

Wheel cart with supplies to sampling location

Put together clothes rack for hanging enteral feeding pouches for gravity filtration and weigh down with bricks. Set up shade structure to cover clothes rack.

Put on fresh gloves

Remove enteral feeding pouch from plastic wrapper

Label pouch with unique sample site/time identifier

Walk pouch to designated sampling site, rinse pouch three times with sampling water, and directly fill pouch with water to 1000 mL mark while keeping the tubing out of the water

Using temperature/salinity probe, record the temperature and salinity at the site the sample was collected

Secure the top of the pouch and return to clothes rack

Using a zip tie, secure the pouch to the top of the clothes rack

Using a small tube cutter, cut the tubing attached to the pouch at the end of the less-rigid opaque section beneath the first purple joint

Put on fresh gloves, and grab one luer lock adapter and a Sterivex filter

Place the barb end of the luer lock adapter into the cut end of the tubing, remove the Sterivex filter, and lock the filter to the tubing

Label the filter with an ethanol-proof lab marker with a unique sample site/time identifier

Using a small bungee cord, secure the filter to the bottom of the clothes rack so that it does not blow in the wind or brush against other filters

Using a flathead screwdriver, twist the inside of the purple connector on the pouch tubing to start the flow of water

Allow water to filter until all water has passed through (1-2 hours), or until drips slow to less than one/minute (clog); if the latter, record the amount of water that has passed through the filter

Remove the filter, and using a sterile 3 mL syringe, pass air through the filter until excess water has been removed

Cap the inlet end of the Sterivex filter with a male-female luer lock cap and the outlet end with a silicone rubber cap

Label a small Whirl-Pak bag with the unique sample site/time and place the filter inside

Place bag on ice until transporting to -15°C freezer at the conclusion of field sampling

If pouches need to be transported while still filtering:

Using a flathead screwdriver, twist the inside of the purple connector to stop the flow of water on any still-filtering bags

Carefully lift the clothes rack on top of the cart; secure with zip ties

Using a flathead screwdriver, twist the inside of the purple connector to start the flow of water on any still-filtering bags. The cart can now be transported as pouches are still filtering.

Transport filters to -15°C freezer to await extraction