Optimized QIAGEN DNeasy Blood & Tissue kit Protocol for Environmental DNA Extraction

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.

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.

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.

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.

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.

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.

Clean bench and material with water, 0.6% sodium hypochlorite solution, and water after use.

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.

Clean bench with water, 0.6% sodium hypochlorite solution, and water before use.

Clean pipettes and centrifuges by wiping them down with water, 0.6% sodium hypochlorite solution, water, and ethanol.

Install all the material on the benchtop including microtubes, reagents and collection tubes from the QIAGEN DNeasy Blood and Tissue Kit, and racks.

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.

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).

Add 450µL of ATL solution to each microtube.

Add 50µL of proteinase K to each microtube.

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

Place microtubes in the thermomixer. Incubate for a minimum of 2h 0m 0s at 56°C with shaking at 900 rpm for lysis of the filtrate.

Note: GF filters should not digest.

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

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.

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.

Add 500µL of AL buffer to each microtube. Change pipette tip between each microtube. Vortex.

Incubate in the thermomixer 0h 10m 0s at 56°C.

Quick spin.

Add 500µL of ethanol 96-100% to each microtube. Change pipette tip between each microtube.

Mix microtubes by inversion for 0h 0m 15s.

Quick spin.

Pipet up to 690µL of the mix into a DNeasy column placed in a 2 mL collection tube.

Centrifuge 0h 1m 0s at 6,000 g. Discard flow-through and collection tube.

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.

Repeat steps 26 to 28 once all the solution went through the column.

Note: DNA is in the column.

Add 500µL of AW1 buffer. Change pipette tip between each microtube.

Centrifuge 0h 1m 0s at 6,000 g. Discard flow-through and collection tube.

Add 500µL of AW2 buffer. Change pipette tip between each microtube.

Centrifuge 0h 3m 0s at 17,500 g. Discard flow-through and collection tube.

Place the DNeasy column in a new collection tube and centrifuge again at 17,500 g for 0h 2m 0s to dry the column.

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

Add 100µL 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.

Incubate at Room temperature for 0h 5m 0s.

Centrifuge 0h 1m 0s at 6,000 g.

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 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 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.

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, 0.6% sodium hypochlorite solution, and water.

Clean bench with water, 0.6% sodium hypochlorite solution, and water.

Rinse microtube racks by spraying them with 0.6% sodium hypochlorite solution, and rinsing them with water.