Optimized Macherey-Nagel NucleoSpin Tissue Protocol for Environmental DNA Extraction

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).

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.

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.

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.

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.

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 0h 0m 30s, and ethanol (i.e., to dry equipment and avoid rust). Change gloves. Repeat steps 3 to 6 for each filter.

Prepare an extraction negative control for each extraction day. Humidify the filter with 250µL of Milli-Q water, add 250µL 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.

Clean bench with water, 0.5% sodium hypochlorite solution, and water or with water, Alconox, 0.5% sodium hypochlorite solution, and ethanol.

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.

Before use, clean bench with water, 0.5% sodium hypochlorite solution, and water or with water, Alconox, 0.5% sodium hypochlorite solution, and ethanol.

Clean pipettes and centrifuges by wiping them down with water, 0.5% sodium hypochlorite solution, water, and ethanol.

Install all the material on the benchtop including microtubes, reagents, collection tubes from the Macherey-Nagel NucleoSpin Tissue kit, and racks.

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).

Prepare a mastermix of 360µL of T1 solution and 50µL 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.

Mix microtubes by inversion for few seconds. Make sure that the filter stays immerged in the solution.

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 . 3h 0m 0s.

Centrifuge microtubes at 18,000 g during 0h 1m 0s.

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.

To avoid leaking issues, transfer the eluate to a new labeled 2 mL microtube.

Add 400µL 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.

Incubate in the thermomixer for 0h 10m 0s at 70°C.

Quick spin.

Add 420µL 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.

Mix microtubes by inversion for few seconds or vortex microtubes for few seconds.

Quick spin.

Pipet up to 650µL of the mix into a MN column placed in a 2 mL collection tube.

Centrifuge 0h 1m 0s 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.

Repeat steps 25 to 26 until all the solution has passed through the column. Change the collection tube.

NOTE: DNA is on the column.

Add 500µL of BW buffer. Change pipette tip between each microtube or use a multidispense pipette with sterile tips, not changing pipette tips.

Centrifuge 0h 1m 0s at 11,000 g. Discard flow-through and collection tube.

Add 600µL of B5 buffer. Change pipette tip between each microtube or use a multidispense pipette with sterile tips, not changing pipette tips.

Centrifuge 0h 1m 0s at 11,000 g. Discard flow-through.

Centrifuge again at 11,000 g for 0h 1m 0s to dry the column.

Place the column into a 2 mL labeled Eppendorf Safe-Lock microtube.

Add 50µL of BE buffer (previously heated at 70°C) on the center of the membrane.

Incubate at Room temperature for 0h 1m 0s.

Centrifuge 0h 1m 0s at 11,000 g.

Repeat steps 34 to 36 for a second round of elution.

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 to limit contamination. We favour the long term storage of DNA extracts at to maximize the preservation of DNA integrity, when no detections are planned. -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.

Throw liquid bench top trash into the liquid trash of the laboratory.

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.

Clean bench with water, 0.5% sodium hypochlorite solution, and water or with water, Alconox, 0.5% sodium hypochlorite solution, and ethanol.