Jun 26, 2026

OceanOmics Centre: eDNA Extraction from Filter Paper using modified Qiagen DNeasy Blood and Tissue Kit

DOI
https://dx.doi.org/10.17504/protocols.io.5jyl8x58dv2w/v1
OceanOmics Centre: eDNA Extraction from Filter Paper using modified Qiagen DNeasy Blood and Tissue Kit
  • 1Minderoo OceanOmics Centre at UWA;
  • 2Minderoo Foundation
Laura Missen
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DOI: https://dx.doi.org/10.17504/protocols.io.5jyl8x58dv2w/v1
External link: https://www.uwa.edu.au/oceans-institute/partnerships/minderoo-oceanomics-centre-at-uwa
Protocol Citation: Laura Missen, Marcelle Ayad, Anna Depiazzi, Georgia Nester, Ebony Thorpe, OceanOmics UWA 2026. OceanOmics Centre: eDNA Extraction from Filter Paper using modified Qiagen DNeasy Blood and Tissue Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8x58dv2w/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
Created: January 06, 2026
Last Modified: June 26, 2026
Protocol  Integer ID: 237128
Keywords: eDNA, environmental DNA, DNA Extraction, DNA extraction from filter paper, marine eDNA, Buffer ATL, Proteinase K digestion, Illumina sequencing, Metabarcoding, Qiagen DNeasy Blood and Tissue Kit , edna extracts from marine environmental dna, marine environmental dna, modified qiagen dneasy blood, purification of total dna, oceanomics centre, qiagen dneasy blood, mixed cellulose ester filter membrane, edna extract, digestion to extraction, purification, total dna, extraction from filter paper, generation sequencing, µl of buffer ae, eluting edna, tissue kit handbook, μl molecular grade ethanol, dna
Abstract
This method is employed by the OceanOmics Centre to generate eDNA extracts from marine Environmental DNA (eDNA) samples for metabarcoding and Next-Generation Sequencing (NGS).

This protocol uses the DNeasy Blood and Tissue Kit (Qiagen) following a modified version of the “Purification of Total DNA from Animal Tissues (Spin-Column Protocol)” (Qiagen DNeasy Blood and Tissue Kit Handbook, pp. 33-36). Modifications include (1) increasing reagent volumes (to 540 μL ATL lysis buffer, 60 μL Proteinase K, 600 μL AL buffer, 600 μL molecular grade ethanol), (2) extending digestion at 56ºC to 3-h, and (3) eluting eDNA twice with 50 µL of Buffer AE (Qiagen), which yields a total elution volume of 100 µL.

This protocol was optimised for 47 mm mixed cellulose ester filter membranes, preserved in Buffer ATL (Qiagen) or CTAB, and yields DNA suitable for high-throughput amplicon sequencing on Illumina platforms. From digestion to extraction, 24 samples will take approximately 6-7 hours.
Guidelines
This protocol is suitable for DNA extraction from 47 mm mixed cellulose membrane filters. The filters should be stored in 2 mL DNA LoBind tubes in 540 µL of preservation buffer. This method has been validated with both buffer ATL (Qiagen) and CTAB (Promega Corporation). If the filter is not stored in buffer, it should be frozen immediately after filtration at -80°C.

Standard lab set-up:
  • Ensure competency and training requirements are met.
  • Wear gloves and a lab coat during extraction process.
  • Use tips with filters to avoid contamination.
  • Wipe workspace with 10% bleach, leave to decontaminate for 10 minutes before wiping with ddH20.
  • Sterilise all equipment (vortex, minispin, pen markers, outside of tip boxes, pipettes) before use.
  • Scissors and tweezers must be sterilised at the start of the protocol and between each filter cutting:

  1. Prepare one 50 mL tube with 10% bleach
  2. Prepare one 50 mL tube of ddH20.
  3. Dip scissors and tweezers in bleach for 10 minutes, then rinse in distilled water. Scissors must be open.
  4. Repeat after each use.

  • UV pipettes, racks and tubes (lids open) for 15 minutes before use.
  • Wipe the inside of the centrifuge and rotor with 70% ethanol.
  • Mop floors with 10% bleach, leave to decontaminate for 10 minutes before mopping with ddH20.
  • Each run requires an extraction blank.
  • Ensure all reagents are aliquoted in appropriate amounts, and stored according to manufacturers' recommendations. Never pipette directly from reagent stocks.
  • All waste must be disposed of following institutional biosafety regulations.
Materials
Equipment
  • Biological scissors and tweezers (optional)
  • Vortex
  • Minispin/Centrifuge
  • Incubator shaker
  • UV sterilisation cabinet

Consumables
  • Eppendorf 2.0 mL DNA LoBind Tubes, PCR Clean
  • Eppendorf 1.5 mL DNA LoBind Tubes, PCR Clean
  • 50 mL Falcon tubes for aliquoting reagents

Reagents
QIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
Buffer ATLQiagenCatalog #19076 Proteinase KQiagenCatalog #19133
100% Molecular grade ethanol Qubit dsDNA HS assay kitFisher ScientificCatalog #Q32854

Safety warnings
This protocol involves the use of potentially hazardous biological materials, chemicals (including ethanol and enzymatic reagents), and electrical laboratory equipment. Always wear appropriate personal protective equipment (PPE), including a lab coat, gloves, and eye protection. Be familiar with the relevant Safety Data Sheets (SDS) and laboratory risk assessments before commencing work. Follow institutional safety guidelines and dispose of waste in accordance with local regulations. Exercise caution when handling sharp instruments, thermal cyclers, and high-speed centrifuges to prevent injury or equipment damage.

General Laboratory Safety:
  • Perform all steps in accordance with your institutions biosafety and chemical safety regulations.
  • Always wear lab coat, gloves, and safety glasses when handling kit components.
  • Dispose of all waste following local hazardous waste disposal.
Chemical Hazards:
  • Buffer ATL and AL contain Guanidine Hydrochloride. This is harmful if swallowed, inhaled, or in contact with skin.
  • Kit contains ethanol and isopropanol. Keep away from ignition sources and flames.
  • Avoid mixing guanidine hydrochloride with bleach or strong oxidiser as toxic gas will form.
Best Practice:
  • Ensure all reagents are stored as recommended in manufacturer's protocol.
  • Conduct in a well-ventilated area or fume hood if available.
Link to SDS document: Download Qiagen DNeasy Blood and Tissue Kit SDS.pdfQiagen DNeasy Blood and Tissue Kit SDS.pdf724.1KB

Before start
  1. Pre-heat incubator shaker to 56 °C .
  2. Buffer AW1 and AW2 are supplied as concentrates. When opening a new DNeasy Blood & Tissue kit, add the appropriate volume of molecular grade ethanol (100%) as indicated on the bottle to obtain a working solution.
  3. Buffer AL may form a precipitate upon storage. Check for precipitate and, if necessary, warm to 55°C to resuspend.
  4. Aliquot the required volume of reagents (ATL, ProK, AL, Ethanol, AW1, AW1, AE) to process your sample batch from the stock tubes into new tubes. This will minimise the possibility of contamination between different sample batches.
  5. Sterilise scissors and tweezers (refer to Guidelines).
  6. Thaw samples at room temperature for 30 minutes.
  7. Label and UV sterilise tubes:
  • Two 2.0 mL DNA LoBind tubes per sample, one for transferring half the filter paper into, the other for transferring the lysate into.
  • One spin-column per sample (available from the Qiagen DNeasy Blood and Tissue Kit; comes with a collection tube).
  • Two 1.5 mL DNA LoBind tubes per sample for DNA extracts (one for an archive stock and one for a working stock).
DNA Digestion
This protocol follows previous sample filtration and preservation steps described in the following protocols:
  • OceanOmics Centre: eDNA Sample Filtration with MasterFlex®.
  • OceanOmics Centre: eDNA Sample Collection with the Ocean Diagnostic Ascension Device.

(Optional) To maximise yield and volume from each sample, filter papers can be cut in half, extracted separately, and then combined once eluted. This requires that you apply the following steps to both the primary (labelled as 'A') and secondary (labelled as 'B') tubes.

Use sterilised scissors to cut the filter paper in half, then use sterilised tweezers to remove one-half of the filter paper and place into a new 2.0 mL labelled (secondary) tube. Sterilise scissors and tweezers between each sample using 10% bleach then rinse with ddH2O (as per the guidelines).
Add 540 µL of Buffer ATL to all the secondary tubes created in step 1.
Using sterilised scissors, and keeping the filter paper in the tube, cut the filter paper into small pieces (<1 cm2) to increase exposed surface area. Ensure that all pieces remain submerged in the buffer after cutting.
Sterilise scissors and tweezers between each sample using 10% bleach then rinse with ddH2O.
Pipette 60 µL of Proteinase K into each sample tube. Change the pipette tip between each sample and pipette directly onto the filter membrane.
Vortex each sample for 15 seconds and spin-down briefly.
Place tubes in a rack and place the rack into the incubator shaker for 3 hours at 300 rpm, 56°C
Once digestion is complete, remove samples from the incubator. This product is referred to as the lysate.

This is a safe stopping point.
DNA can be stored at -20°C long-term. Avoid freeze/thaw cycles since this can degrade DNA.
Transfer the liquid from each tube into a new labelled 2 mL DNA LoBind tube. Use the pipette tip to squeeze the filter paper to the side of the tube to obtain as much liquid as possible. Repeat as many times as necessary for each sample.

This is a safe stopping point.
DNA can be stored at -20°C long-term. Avoid freeze/thaw cycles since this can degrade DNA.
DNA Extraction
If frozen, remove samples from the freezer and allow to thaw at room temperature for 30 minutes.
Vortex and spin-down samples.
Add 600 µL of Buffer AL to each sample.

NOTE: buffer AL may form a precipitate upon storage, if present, heat and mix until resuspended. Buffer AL is viscous and bubbly, change tips between samples.
Vortex and spin-down.
Incubate on the bench top at Room temperature for 10 minutes.

Add 600 µL of 100% molecular grade ethanol to each sample.
Vortex and spin-down.
Add 650 µL of lysate into the correspondingly labelled spin-column.
Centrifuge at 8000 x g for 1 minute.

After centrifuging, the DNA will be bound to the silica membrane in the spin-column and the remainder of your sample (i.e., flow-through) will pass through the spin-column into the collection tube.

Remove the spin-column from the collection tube and discard the flow-through into a designated waste container. Carefully dry the mouth end of the collection tube onto a Kimwipe then place the spin-column back into the collection tube.
Repeat steps 17-19 until all the lysate has passed through the spin-column. This should be a total of three repetitions.
Add 500 µL of Buffer AW1 to the spin-column.
Centrifuge at 8000 x g for 1 minute.

Place the spin-column into a new collection tube. Discard the previously used collection tube together with the flow-through.
Add 500 µL of Buffer AW2 to the spin-column.
Centrifuge at 10000 x g for 3 minutes.

Transfer the spin-column into a pre-labelled 1.5 mL DNA LoBind tube. This is the working stock tube. Discard the previously used collection tube together with the flow-through.
Carefully pipette 50 µL of Buffer AE directly onto the silica membrane of the spin-column without touching the membrane itself with the pipette tip.
Incubate on the bench top at Room temperature for 15 minutes with the spin-column lid closed.

Centrifuge at 10000 x g for 1 minute.

Repeat steps 27-29 so the final elution volume is 100 µL.
If no duplicates exist per sample, pipette mix, then transfer 50 µL of eluted DNA into the pre-labelled 1.5 mL LoBind tube for archiving (i.e., archive stock). The remaining 50 µL of eluted DNA is the working stock.
If duplicates exist per sample (i.e. have ‘A’ and B’ per sample number):
a. Take the full volume (100 µL) of eluted DNA from tube A (working stock) and add to tube B (archive stock). Tube B will temporarily hold 200 µL. b. Using a 200 µL pipette, pipette mix tube B 10 times. c. Transfer 100 µL from tube B back to tube A. This will ensure your DNA sample is homogenised between working and archive stocks.
This is a safe stopping point.
The working stock should be stored at 4°C for immediate processing. If not being processed immediately, or after being processed, the working stock should be stored at -20°C. The archive stock should be stored at -80°C.
DNA Quantification
Quantify 2 µL of each sample using Qubit HS dsDNA kit.
If proceeding with eDNA metabarcoding, follow the steps described in OceanOmics Centre: 12S, 16S and COI Metabarcoding of Marine Vertebrates.

Protocol references