Nov 18, 2023
Version 1
eDNA Water Sample Collection, Preservation and Extraction (low-tech sampling, modified Qiagen PowerWater extraction) V.1
Forked from QIAGEN DNeasy Power Water SOP
- 1University of Arizona
Protocol Citation: Eldridge Wisely 2023. eDNA Water Sample Collection, Preservation and Extraction (low-tech sampling, modified Qiagen PowerWater extraction). protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22b7pl1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working. In the future, we would like to get an automated sampler, to reduce potential DNA contamination sources in the field, but this protocol works well when appropriate field negative controls are included in the analysis.
Created: November 18, 2023
Last Modified: November 18, 2023
Protocol Integer ID: 91143
Keywords: marine environmental dna, water sample collection, environmental dna, galápagos fieldwork, extraction protocol, edna, qiagen powerwater extraction, dna, gulf of california fieldwork, extraction, marine, preservation
Abstract
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.
Materials
Equipment
Reusable Filter Unit
NAME
Filter unit
TYPE
Nalgene
BRAND
NAL300-4100
SKU
LINK
Equipment
Oil-less vacuum pump
NAME
vacuum pump
TYPE
Fristaden Lab
BRAND
VP-10L
SKU
LINK
Equipment
Wide-mouth HDPE bottle
NAME
sample collection bottle
TYPE
Nalgene
BRAND
N311-1000BPC
SKU
LINK
Zymo DNA/RNA ShieldFisher ScientificCatalog #50-125-1706
Equipment
HAWP MF-Millipore Membrane Filter, 0.45 µm pore size
NAME
Membrane filter
TYPE
Millipore
BRAND
HAWP04700
SKU
LINK
0.45 um 47 mm
SPECIFICATIONS
Equipment
PowerWater DNA bead tube
NAME
Tube
TYPE
Qiagen
BRAND
14900-50-NF-BT
SKU
Equipment
1.5mL sterile tubes
NAME
1.5 mL tube
TYPE
Axygen
BRAND
MCT150CS
SKU
Qiagen PowerWater kitQiagenCatalog #14900-50-NF
Equipment
vortex adapter for 5mL tubes
NAME
vortex adapter
TYPE
Qiagen
BRAND
13000-V1-5
SKU
LINK
Protocol materials
10% Bleach
Zymo DNA/RNA ShieldFisher ScientificCatalog #50-125-1706
Qiagen PowerWater kitQiagenCatalog #14900-50-NF
DNA/RNA ShieldZymo ResearchCatalog #R1100-50
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).
Water collection and filtration
23m
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.
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).
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.
Clean the reusable filter funnel and included filter support pedestal with 10% Bleach and rinse 3x with remove the upper portion of the reusable filter funnel,
Rinse 3x with purified water (drinking water if available, tap water if purified is not available or is cost prohibitive)
Assemble the reusable filter funnel with the 0.45 micron MCE filter inside it.
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.
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 DNA/RNA ShieldZymo ResearchCatalog #R1100-50 to the filter so that the filter is covered by the buffer, let the buffer sit on the filter for at least 00:00:05 , then re-apply the vacuum again briefly, until the liquid has been pulled through the filter.
5s
Remove the filter funnel portion of the reusable filter unit so that the filter is exposed.
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.
for each subsequent liter of water to be processed,
Clean the sample bottle (1L Nalgene bottle)
Equipment
Wide-mouth HDPE bottle
NAME
sample collection bottle
TYPE
Nalgene
BRAND
N311-1000BPC
SKU
LINK
with 10% Bleach 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.
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.
eDNA Extraction
23m
Heat Solutions PW1 and PW3 to 55 °C and Buffer EB to 70 °C .
Add 1 mL of Solution PW1 to the PowerWater DNA Bead Tube.
Secure the tube horizontally to a
Equipment
vortex adapter for 5mL tubes
NAME
vortex adapter
TYPE
Qiagen
BRAND
13000-V1-5
SKU
LINK
.
Vortex at maximum speed for 00:05:00 .
5m
Transfer the supernatant to a clean 2 mL Collection Tube (provided). Draw up the supernatant using a 1 mL pipette 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.)
Centrifuge at 13000 x g, Room temperature, 00:01:00
1m
Avoiding the pellet, transfer the supernatant to a clean 2 mL Collection Tube (provided).
Add 200 µL of Solution IRS and vortex briefly to mix. Incubate at 2-8 °C for 00:05:00 .
5m
Centrifuge the tubes at 13000 x g, Room temperature, 00:01:00 .
1m
Avoiding the pellet, transfer the supernatant to a clean 2 ml 2 mL Collection Tube (provided).
Carefully, add 650 µL of Solution PW3 and thoroughly pipette mix or vortex briefly to mix.
Load 650 μl of supernatant onto an MB Spin Column. Centrifuge at 13000 x g, Room temperature, 00:01:00 . Discard the flow-through. Repeat until all the supernatant has been processed.
1m
Place the MB Spin Column Filter into a clean 2 mL Collection Tube (provided).
Add 650 µL of Solution PW4 (shake before use). Centrifuge at 13000 x g, Room temperature, 00:01:00 .
1m
Discard the flow-through and add 650 µL of ethanol (provided) and centrifuge at 13000 x g, Room temperature, 00:01:00 .
1m
Discard the flow-through and centrifuge again at 13000 x g, Room temperature, 00:02:00 .
2m
Place the MB Spin Column into a clean 2 mL Collection Tube (provided).
Add 100 µL of 70 °C Solution EB to the center of the white filter membrane.
Incubate at Room temperature for a minimum of 00:10:00
10m
Centrifuge at 13000 x g, Room temperature, 00:01:00 .
1m
Discard the MB Spin Column, and place eluted liquid containing the eDNA into a new 1.5mL DNAse- , RNAse-free, sterile 1.5mL tube.
Equipment
1.5mL sterile tubes
NAME
1.5 mL tube
TYPE
Axygen
BRAND
MCT150CS
SKU
The DNA is now ready for downstream applications.
QIAgen recommend storing DNA frozen ( -90 °C to -15 °C ) as Solution EB does not contain EDTA