TITLE: Optimized Macherey-Nagel NucleoSpin Tissue Protocol for Environmental DNA Extraction
AUTHORS: Michaela Harris, Jade Larivière, Valérie Belliveau, Marion Chevrinais, Laury-Ann Dumoulin, Francis LeBlanc, Cloé Lepage, Nellie Gagné, 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]
Wash Buffer B5 is supplied as concentrated solution. Before using for the first time, add the appropriate amount of ethanol (95–100%) as indicated on the bottle to obtain a working solution. Wash Buffer B5 can be stored at 15 – 25 °C for at least one year.

If proteinase K is solid, add the indicated volume of proteinase Buffer PB to dissolve lyophilized proteinase K. Proteinase K solution is stable at -20 °C for at least 6 months.

Preheat a thermomixer at 56°C and an another one at 70°C .

[GUIDELINES]
The extraction of environmental DNA should be processed in a dedicated room (not the same room as tissue DNA extraction) with filtered air and positive air pressure. All the samples, consumables and material entering the room should be cleaned with 0.5% sodium hypochlorite solution. Laboratory users should be trained to work in clean conditions (specific instructions about when to wear and change sterile gloves, coats, mobcaps, chirurgical masks, and overshoe protections). The extraction room should be decontaminated between projects or every week with a 0.5% 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, weigh boats, waste beaker, microtube opener, and gloves. 
SECTION: DNA Extraction
10. Before use, clean bench with water, 0.5% sodium hypochlorite solution, and water or with water, Alconox, 0.5% sodium hypochlorite solution, and ethanol. 
SECTION: Filter preparation
3. Above a clean weigh boat, take a filter with clean tweezers, unfold it, and cut it in two equal halfs with clean scissors. Alternatively, use sterilized and disposable toothpicks and scalpels to transfer and cut filters in a weigh boat. Because filters were preserved with ethanol 95-100%, leave one half to air dry on the weigh boat. 
Note: Try to divide the filtrate equally on each half. 
SECTION: Filter preparation
4. Once air dried, roll the filter half (filtrate inside), and put it in the extraction microtube (2 mL microtube with a Lyse&Spin basket) using clean tweezers (or toothpicks and scalpel). If DNA extraction will occur on 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 of the filter with tweezers, put it into the Eppendorf Safe-Lock 2 mL previously identified, and store it as back up at -80 °C. Alternatively, store the second half filter in its original tubes containing ethanol 95-100% at -20 °C.

SECTION: Filter preparation
6. If not using disposable equipment, rinse tweezers, scissors, and weigh boats with water, 0.5% sodium hypochlorite solution, and water for final rinse during at least 30 s, and ethanol (i.e., to dry equipment and avoid rust). Change gloves. Repeat steps 3 to 6 for each filter.

SECTION: Critical Notes for DNA Extraction
9. DO NOT TOUCH microtube edges with gloves while opening. Always use a microtube opener. In case of doubt for contamination, change gloves before touching another sample. 

DO NOT OPEN more than one microtube at a time to limit contamination. Alternatively, if using a multidispense pipette, leave space between opened tubes. 

ALWAYS do a quick spin before microtube opening to limit aerosols. In case of doubt for contamination, rinse the microtube opener with water, 0.5% sodium hypochlorite solution, and water. 

Optimally, change the pipette tip between microtubes when adding a solution, even if the same solution is added. Alternatively, use a multidispense pipette with sterile tips, not changing pipette tips while adding the same solution.
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. Prepare a mastermix of 360 µL of T1 solution and 50 µL of proteinase K to dispense to each microtube.
NOTE: Work as quickly as possible once proteinase K is mixed to T1 to avoid self digestion.
SECTION: DNA Extraction
15. Mix microtubes by inversion for few seconds. Make sure that the filter stays immerged in the solution. 
SECTION: DNA Extraction
16. Place microtubes in the thermomixer. Incubate   at 56 °C with shaking at 900 rpm  to digest the filtrate. 
NOTE: GF filters should not digest. 
NOTE: If not overnight, incubation should last at least 180 min. 

SECTION: DNA Extraction
17. Centrifuge microtubes at 18,000 g during 1 min.
NOTE: If lysis solution is still present in the column after centrifugation, redo step 17 until all the solution went through the column. Make note in an excel sheet.
SECTION: DNA Extraction
18. To avoid leaking issues, transfer the eluate to a new labeled 2 mL microtube.
SECTION: DNA Extraction
19. Add 400 µL of B3 buffer to each microtube. Change pipette tip between each microtube or use a multidispense pipette with sterile tips, not changing pipette tips.  Mix microtubes by inversion for few seconds or vortex microtubes for few seconds. 
SECTION: DNA Extraction
20. Incubate in the thermomixer for 10 min at 70 °C.
SECTION: DNA Extraction
21. Quick spin.
SECTION: DNA Extraction
22. Add 420 µL of ethanol 95-100% to each microtube. Change pipette tip between each microtube or use a multidispense pipette with sterile tips, not changing pipette tips. 
SECTION: DNA Extraction
23. Mix microtubes by inversion for few seconds or vortex microtubes for few seconds. 
SECTION: DNA Extraction
24. Quick spin.
SECTION: DNA Extraction
25. Pipet up to 650 µL  of the mix into a MN column placed in a 2 mL collection tube.

SECTION: DNA Extraction
26. Centrifuge 1 min at 11,000 g. Discard flow-through. 
NOTE: When flow-through is discarded in the trash, be careful not to contaminate gloves and bench top.
SECTION: DNA Extraction
27. Repeat steps 25 to 26 until all the solution has passed through the column. Change the collection tube. 
NOTE: DNA is on the column.
SECTION: DNA Extraction
28. Add 500 µL of BW buffer. Change pipette tip between each microtube or use a multidispense pipette with sterile tips, not changing pipette tips. 
SECTION: DNA Extraction
29. Centrifuge 1 min at 11,000 g. Discard flow-through and collection tube.
SECTION: DNA Extraction
30. Add 600 µL of B5 buffer. Change pipette tip between each microtube or use a multidispense pipette with sterile tips, not changing pipette tips. 
SECTION: DNA Extraction
31. Centrifuge 1 min at 11,000 g. Discard flow-through.
SECTION: DNA Extraction
32. Centrifuge again at 11,000 g for 1 min to dry the column. 
SECTION: DNA Extraction
33. Place the column into a 2 mL labeled Eppendorf Safe-Lock microtube.
SECTION: DNA Extraction
34. Add 50 µL of BE buffer (previously heated at 70 °C) on the center of the membrane. 

SECTION: DNA Extraction
35. Incubate at Room temperature for 1 min.
SECTION: DNA Extraction
37. Repeat steps 34 to 36 for a second round of elution. 
SECTION: DNA Extraction
38. Discard the column and store the 2 mL labeled Eppendorf Safe-Lock microtube with the eluate at4 °C  for short-term storage, at -20 °C for medium-term storage, and at -80 °C  for long-term storage.
NOTE: The storage protocol of DNA extracts maximizes 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: Filter preparation
1. Clean bench before use with water (i.e., Milli-Q water, hereafter "water" for cleaning procedures, to remove Macherey-Nagel solutions with guanidine salts prior bleaching; see warnings for details), 0.5% sodium hypochlorite solution (i.e., to degrade DNA), and water (i.e. to rinse traces of sodium hypochlorite). Alternatively, clean bench before use with Alconox, 0.5% sodium hypochlorite solution and ethanol 70% (hereafter referred to ethanol for cleaning procedures). 
SECTION: Filter preparation
8. Clean bench with water, 0.5% sodium hypochlorite solution, and water or with water, Alconox, 0.5% sodium hypochlorite solution, and ethanol. 
SECTION: DNA Extraction
11. Clean pipettes and centrifuges by wiping them down with water, 0.5% sodium hypochlorite solution, water, and ethanol.
SECTION: DNA Extraction
12. Install all the material on the benchtop including microtubes, reagents, collection tubes from the Macherey-Nagel NucleoSpin Tissue kit, and racks.
SECTION: DNA Extraction
36. Centrifuge 1 min at 11,000 g.
SECTION: DNA Extraction
39. Throw liquid bench top trash into the liquid trash of the laboratory. 
SECTION: DNA Extraction
40. Rinse the liquid bench top trash with water, discard this water in the liquid trash of the laboratory, then clean it with water, 0.5% sodium hypochlorite solution, and water.
SECTION: DNA Extraction
41. Clean bench with water, 0.5% sodium hypochlorite solution, and water or with water, Alconox, 0.5% sodium hypochlorite solution, and ethanol. 
SECTION: Filter preparation
7. Prepare an extraction negative control for each extraction day. Humidify the filter with 250 µL of Milli-Q water, add 250 µL of ethanol 95-100%, and proceed with steps 3 and 4. Alternatively, the extraction negative control could be prepared at step 14 by adding T1 solution only.
Note 1: Use a filter of material and porosity identical to those from the project.