TITLE: eDNA Water Sample Collection, Preservation and Extraction (low-tech sampling, modified Qiagen PowerWater extraction)
AUTHORS: Eldridge Wisely

[DESCRIPTION]
Marine environmental DNA (eDNA) collection, filtration, preservation, and extraction protocol used in Galápagos fieldwork 2021-2023, and Gulf of California fieldwork in 2023 by Eldridge Wisely.

[BEFORE_START]
Solution PW1 must be warmed at 55°C for 5–10 minutes to dissolve precipitates prior to use. 
Solution PW1 should be used while still warm.
Assume Solution PW3 has precipitated, and preemptively  heat at 55°C for 5–10 minutes to dissolve precipitate.
Shake to mix Solution PW4 before use.
Perform all centrifugation steps at room temperature (15–25°C).

[STEPS]
SECTION: Water collection and filtration
1. Collect water samples in a cleaned (see step 12) 1L Nalgene bottle by submerging the bottle and then opening it and closing it underwater. This step can be adapted to your sampling logistics, while reducing as much as possible any contact between your skin or clothes or other equipment with the water being sampled.  Label your samples, and take metadata including latitude and longitude  , water temperature, and any other variables you would like to have for later analysis.  

SECTION: Water collection and filtration
4. Clean the reusable filter funnel and included filter support pedestal with   and rinse 3x with remove the upper portion of the reusable filter funnel, 

SECTION: Water collection and filtration
10. Using two sets of sterile forceps (tweezers cleaned with bleach solution and then 70% EtOH, and allowed to air dry), pick up the white filter membrane at opposite edges and roll the filter into a cylinder with the top side facing inward.
SECTION: eDNA Extraction
15. Add 1 mL  of Solution PW1 to the PowerWater DNA Bead Tube.

SECTION: eDNA Extraction
16. Secure the tube horizontally to a 
 
.
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17. Vortex at maximum speed for  5 min. 

SECTION: eDNA Extraction
18. Transfer the supernatant to a clean  2 mL  Collection Tube (provided). Draw up the supernatant using a 1 mLpipette tip by placing it down into the beads. (Note: Placing the pipette tip down into the beads is required. Pipette until you have removed all the supernatant. Expect to recover  650-800 µL of supernatant.)

SECTION: eDNA Extraction
19. Centrifuge at 13000 x g, 1 min 

SECTION: eDNA Extraction
20. Avoiding the pellet, transfer the supernatant to a clean 2 mL  Collection Tube (provided).

SECTION: eDNA Extraction
21. Add  200 µL  of Solution IRS and vortex briefly to mix. Incubate at 2-8 °C  for  5 min .

SECTION: eDNA Extraction
22. Centrifuge the tubes at 13000 x g, 1 min .

SECTION: eDNA Extraction
23. Avoiding the pellet, transfer the supernatant to a clean 2 ml 2 mL  Collection Tube (provided).

SECTION: eDNA Extraction
24. Carefully, add  650 µL of Solution PW3 and thoroughly pipette mix or vortex briefly to mix.

SECTION: eDNA Extraction
25. Load 650 μl of supernatant onto an MB Spin Column. Centrifuge at 13000 x g, 1 min . Discard the flow-through. Repeat until all the supernatant has been processed.

SECTION: eDNA Extraction
26. Place the MB Spin Column Filter into a clean 2 mL  Collection Tube (provided).

SECTION: eDNA Extraction
27. Add 650 µL  of Solution PW4 (shake before use). Centrifuge at 13000 x g, 1 min .

SECTION: eDNA Extraction
28. Discard the flow-through and add 650 µL of ethanol (provided) and centrifuge at 13000 x g, 1 min.

SECTION: eDNA Extraction
29. Discard the flow-through and centrifuge again at 13000 x g, 2 min.

SECTION: eDNA Extraction
30. Place the MB Spin Column into a clean 2 mL  Collection Tube (provided).

SECTION: eDNA Extraction
31. Add  100 µL  of 70 °C Solution EB to the center of the white filter membrane.

SECTION: eDNA Extraction
32. Centrifuge at 13000 x g, 1 min.

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33. Discard the MB Spin Column, and place eluted liquid containing the eDNA into a new 1.5mL DNAse- , RNAse-free, sterile 1.5mL tube.
 
The DNA is now ready for downstream applications.
SECTION: eDNA Extraction
33.1. QIAgen recommend storing DNA frozen ( -90 °C  to -15 °C ) as Solution EB does not contain EDTA

SECTION: Water collection and filtration
6. Assemble the reusable filter funnel with the 0.45 micron MCE filter inside it.
SECTION: Water collection and filtration
7. Filter the water sample by pouring the contents of the 1 L  Nalgene bottle into the reusable filter unit, while applying a vacuum with the vacuum pump.
SECTION: Water collection and filtration
5. Rinse 3x with purified water (drinking water if available, tap water if purified is not available or is cost prohibitive)

SECTION: Water collection and filtration
8. After sample has been filtered, stop the vacuum and release the pressure by loosening the tubing to the reusable filter unit, then add 500uL of    to the filter so that the filter is covered by the buffer, let the buffer sit on the filter for at least 5 s , then re-apply the vacuum again briefly, until the liquid has been pulled through the filter.

SECTION: Water collection and filtration
9. Remove the filter funnel portion of the reusable filter unit so that the filter is exposed.
SECTION: Water collection and filtration
3. Wear lab gloves for all subsequent steps, and change your gloves if any contact with your sample water occurs, so you don't cross contaminate your samples during the filtration step.
SECTION: Water collection and filtration
11. for each subsequent liter of water to be processed,   

SECTION: Water collection and filtration
13. Each time sampling logistics change, or rinse water availability changes, take a sample of 1L of rinse water and process according to this protocol, as a laboratory negative control.  Also, take a field negative control by leaving rinse water in one of the Nalgene bottles after cleaning and take it to the field with the rest of the bottles and handle the same as the sampling bottles, except don't change the water in the bottles, as a negative field control sample.
SECTION: eDNA Extraction
14. Heat Solutions PW1 and PW3 to 55 °C and Buffer EB to 70 °C . 

SECTION: eDNA Extraction
31.1. Incubate at Room temperature for a minimum of 10 min 

SECTION: Water collection and filtration
2. Store the sample bottles in a cooler on ice  with icepacks to keep them cool and protected from UV light until they can be filtered (within 12 hours of collection).
SECTION: Water collection and filtration
12. Clean the sample bottle (1L Nalgene bottle)
 
with   and rinse 3x with purified water (drinking water if possible). 

Take at least one negative control of the rinse water as described in step 13, in accordance with your sampling and processing logistics and budget.