TITLE: Optimized QIAGEN DNeasy Blood &amp; Tissue kit Protocol for Environmental DNA Extraction
AUTHORS: Marion Chevrinais, Jade Larivière, Laury-Ann Dumoulin, Ariane Therien, Grégoire Cortial, Cloé Lepage, Eric Parent, Geneviève J. Parent

[DESCRIPTION]
This document aims at providing a transparent method and detailing mandatory steps to produce reproducible 1)  preparation of an eDNA filter, and 2) environmental DNA extraction.

[BEFORE_START]
Buffer AL may form a precipitate upon storage. If necessary, warm to 56°C until the precipitate has fully dissolved. 

Buffer AW1 and Buffer AW2 are supplied as concentrated solutions. Before using for the first time, add the appropriate amount of ethanol (96–100%) as indicated on the bottle to obtain a working solution. 

Preheat a thermomixer at 56°C.

[GUIDELINES]
The extraction of environmental DNA (eDNA) should be processed in the dedicated room of the ultraclean laboratory (i.e., with filtered air and positive air pressure) of the Maurice Lamontagne Institute, Fisheries and Oceans Canada. All samples, consumables, and material entering the room are cleaned with a 0.6% sodium hypochlorite solution. Laboratory users are trained to work in clean conditions (i.e., specific instructions about when to wear and change sterile gloves, coats, mobcaps, chirurgical masks, and overshoe protections) according to the SOP 020901_VXX. The extraction room is decontaminated between projects or every week (i.e., if a room is used for a project longer than a week) with a 0.6% sodium hypochlorite solution.

[STEPS]
SECTION: Filter Preparation
2. Install all the material on the benchtop including 2 mL microtubes with Lyse&Spin baskets for extraction, pre-identified 2 mL Eppendorf Safe-Lock for back up, tweezers, scissors, nacelles, waste beaker, microtube opener, and gloves. 
SECTION: Filter Preparation
3. Above a clean nacelle, take a filter with clean tweezers, unfold it, and cut it in half with clean scissors.
Note: Attemps to divide the filtrate equally on each half.
SECTION: Filter Preparation
4. Roll the filter half (filtrate inside) and put it in the extraction microtube (2 mL microtube with a Lyse&Spin basket) using clean tweezers. If DNA extraction will occur a subsequent day, put the filter half into a 2 mL microtube and store it at -20 °C. If DNA extraction will occur on the same day, keep it at room temperature on the bench.
SECTION: Filter Preparation
5. Roll the second half with tweezers, put it in a Eppendorf Safe-Lock 2 mL previously identified, and store it as a back up at -80 °C.
SECTION: Filter Preparation
6. Rinse tweezers, scissors, and nacelle with water, 0.6% hypochlorite bleach, water, and ethanol 96-100% (i.e., to dry equipment and avoid rust; hereafter, ethanol for cleaning procedures). Change gloves. Repeat steps 3 to 6 for each filter.
SECTION: Critical Notes for DNA Extraction
8. DO NOT TOUCH microtube edges with gloves while opening. Always use a microtube opener. In case of doubt for contamination, change gloves. 

DO NOT OPEN more than one microtube at a time to limit contamination. 

ALWAYS do a quick spin before microtube opening to limit aerosols. In case of doubt for contamination, rinse the microtube opener with water, 0.6% sodium hypochlorite, and water. 

ALWAYS change the pipette tip between microtubes when adding a solution, even if the same solution is added. 
SECTION: DNA Extraction
13. Change gloves and work under extractor hood or arm for all subsequent steps because of proteinase K (i.e., may cause breathing difficulties if inhaled).
SECTION: DNA Extraction
14. Add 450 µL of ATL solution to each microtube.

SECTION: DNA Extraction
15. Add 50 µL of proteinase K to each microtube. 

SECTION: DNA Extraction
16. Mix microtubes by inversion for few seconds. Make sure that the filter stays immerged in the solution. 
SECTION: DNA Extraction
17. Place microtubes in the thermomixer. Incubate for a minimum of 120 min at 56 °C with shaking at 900 rpm for lysis of the filtrate.
Note: GF filters should not digest. 
SECTION: DNA Extraction
18. Centrifuge microtubes at 18,000 g during 1 min.
Note: If lysis solution is still present in the column after centrifugation, redo step 18 until all the solution went through the column. Make note in the excel sheet.
SECTION: DNA Extraction
19. Transfer the eluate to a new labeled 2 mL microtube.
Note: The microtube used with the Lysis&Spin basket was often leaking in the centrifuge at next steps which is why changing the microtube at this step is desirable.
SECTION: DNA Extraction
20. Add 500 µL of AL buffer to each microtube. Change pipette tip between each microtube. Vortex. 

SECTION: DNA Extraction
21. Incubate in the thermomixer 10 min at 56 °C.

SECTION: DNA Extraction
22. Quick spin.
SECTION: DNA Extraction
23. Add 500 µL of ethanol 96-100% to each microtube. Change pipette tip between each microtube.

SECTION: DNA Extraction
24. Mix microtubes by inversion for 15 s.

SECTION: DNA Extraction
25. Quick spin.
SECTION: DNA Extraction
26. Pipet up to 690 µL of the mix into a DNeasy column placed in a 2 mL collection tube.

SECTION: DNA Extraction
27. Centrifuge 1 min at 6,000 g. Discard flow-through and collection tube.

SECTION: DNA Extraction
28. Place the DNeasy column in a new collection tube. 
Note: When collection tubes are discarded in the trash, be careful not to contaminate gloves and bench top.
SECTION: DNA Extraction
29. Repeat steps 26 to 28 once all the solution went through the column. 
Note: DNA is in the column.
SECTION: DNA Extraction
30. Add 500 µL of AW1 buffer. Change pipette tip between each microtube.

SECTION: DNA Extraction
31. Centrifuge 1 min at 6,000 g. Discard flow-through and collection tube.

SECTION: DNA Extraction
32. Add 500 µL of AW2 buffer. Change pipette tip between each microtube.

SECTION: DNA Extraction
33. Centrifuge 3 min at 17,500 g. Discard flow-through and collection tube.

SECTION: DNA Extraction
34. Place the DNeasy column in a new collection tube and centrifuge again at 17,500 g for 2 min to dry the column. 

SECTION: DNA Extraction
35. Place the column into a 2 mL labeled Eppendorf Safe-Lock microtube.
SECTION: DNA Extraction
36. Add 100 µL of EB buffer directly in the center of the membrane. 
Note: We use EB buffer instead of the AE buffer provided in the Blood and Tissue kit as EB buffer is a TRIS buffer without EDTA. For rare species detection, we observed qPCR inhibition due to this small concentration of EDTA in the past.
SECTION: DNA Extraction
37. Incubate at Room temperature for 5 min.

SECTION: DNA Extraction
39. Discard the column and store the 2 mL labeled Eppendorf Safe-Lock microtube with the eluate at 4 °C for eDNA detections within two weeks, at -20 °C for eDNA detections between 2 weeks and 3 months, and at -80 °C for eDNA detections later than 3 months.
Note: The storage protocol of DNA extracts is based on the duration prior the DNA detection step and the location of freezers at Maurice Lamontagne Institute. We favour the storage of DNA extracts at  4 °C within the ultraclean laboratory to limit contamination and to maximize chances of eDNA detections since freeze-thaw cycles limit detection of rare molecules (i.e., most likely due to deterioration of DNA's integrity). We favour the storage of DNA extracts within the ultraclean laboratory at -20 °C to limit contamination. We favour the long term storage of DNA extracts at  -80 °C to maximize the preservation of DNA integrity, when no detections are planned.
SECTION: DNA Extraction
40. Throw liquid bench top trash into the liquid trash of the laboratory. 

SECTION: DNA Extraction
9. Clean bench with water, 0.6% sodium hypochlorite solution, and water before use. 
SECTION: DNA Extraction
12. Prepare an extraction negative control for each extraction day. 
Note 1: Use a filter of material and porosity identical to those from the project.
Note 2: If filters were frozen (i.e., usually moisten), then humidify the filter with Milli-Q water and proceed with steps 3 and 4. If filters were in silica beads then proceed with steps 3 and 4 without humidifying. 
SECTION: DNA Extraction
41. Rinse the liquid bench top trash with water, discard this water in the liquid trash of the laboratory, then clean it with water, 0.6% sodium hypochlorite solution, and water.

SECTION: DNA Extraction
11. Install all the material on the benchtop including microtubes, reagents and collection tubes from the QIAGEN DNeasy Blood and Tissue Kit, and racks.
SECTION: DNA Extraction
10. Clean pipettes and centrifuges by wiping them down with water, 0.6% sodium hypochlorite solution, water, and ethanol.
SECTION: DNA Extraction
42. Clean bench with water, 0.6% sodium hypochlorite solution, and water.
SECTION: DNA Extraction
43. Rinse microtube racks by spraying them with 0.6% sodium hypochlorite solution, and rinsing them with water.   
SECTION: Filter Preparation
1. Clean bench with water (i.e., Milli-Q water, hereafter water for cleaning procedures, to remove QIAGEN solutions with guanidine salts prior bleaching; see warnings for details), 0.6% sodium hypochlorite solution (i.e., to degrade DNA), and water (i.e. to rinse traces of sodium hypochlorite) before use. 
SECTION: Filter Preparation
7. Clean bench and material with water, 0.6% sodium hypochlorite solution, and water after use. 
SECTION: DNA Extraction
38. Centrifuge 1 min at 6,000 g.