Apr 07, 2026
A Simplified eDNA Extraction Protocol from Sterivex Filters Using the Qiagen DNeasy Blood & Tissue Kit
- Igor A. Nascimento1,
- Gabriel Antônio Mendes1,
- Heron Hilário1,
- Julia Affonseca1,
- Daniel Cardoso de Carvalho 1,
- Jeniffer Teles1
- 1Conservation Genetics Laboratory - Pontifícia Universidade Católica de Minas Gerais (PUC Minas)
Protocol Citation: Igor A. Nascimento, Gabriel Antônio Mendes, Heron Hilário, Julia Affonseca, Daniel Cardoso de Carvalho , Jeniffer Teles 2026. A Simplified eDNA Extraction Protocol from Sterivex Filters Using the Qiagen DNeasy Blood & Tissue Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp763pgzp/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: April 07, 2026
Last Modified: April 07, 2026
Protocol Integer ID: 314661
Keywords: edna extraction protocol from sterivex filter, extracting environmental dna, simplified edna extraction protocol, environmental dna, reliable biodiversity detection in aquatic ecosystem, quality dna suitable for downstream application, sterivex filter, supporting reliable biodiversity detection, aquatic ecosystem, quality dna, qiagen dneasy blood, dna, strict contamination control procedure, tissue kit this protocol
Abstract
This protocol presents a simplified workflow for extracting environmental DNA (eDNA) from Sterivex filters using the Qiagen DNeasy Blood & Tissue Kit, with adaptations to improve practicality and reproducibility in laboratory routines. The method includes extended lysis, optimized reagent volumes, and strict contamination control procedures.
The protocol is expected to yield high-quality DNA suitable for downstream applications such as PCR and metabarcoding, supporting reliable biodiversity detection in aquatic ecosystems. Its standardized approach facilitates its application in ecological studies, monitoring programs, and large-scale projects.
Guidelines
24.7 Discard the spin column.
24.8 Store the eluted DNA at –20 °C.
Materials
- 1000 µL pipette tips (that fit the inlet of the Sterivex)
- 200 µL pipette tips
- Proteinase K (Qiagen DNeasy Blood 26 Tissue Kit)
- Buffer ATL (Qiagen DNeasy Blood 26 Tissue Kit)
- 1.5/2 mL LoBind tubes
- 5 mL LoBind tubes
- DNeasy Mini spin columns
- Collection tubes
- Sterile 3 mL syringes
- Buffer AL
- Buffer AW1
- Buffer AW2
Safety warnings
Extraction replicates should be processed only from the same sampling site at a time. Do not process samples from different sites simultaneously to avoid cross-contamination.
Before start
If processing a different number of filters, consider the subsequent processing steps (especially centrifugation) when deciding how many filters to extract at once.
Day 1 – DNA Lysis
Clean the bench area and chairs first with 5% bleach, rinse with water to remove bleach residues, and then disinfect with 70% ethanol. Apply RNase Away to all equipment that will be used.
Turn on the incubator and set it to 56 °C.
Wipe down 1000 µL and 200 µL pipettes with RNase Away and expose them to UV light for 15 minutes on each side.
Assemble materials and reagents:
- 1000 µL pipette tips (that fit the inlet of the Sterivex)
- 200 µL pipette tips
- Proteinase K (Qiagen DNeasy Blood 26 Tissue Kit)
- Buffer ATL (Qiagen DNeasy Blood 26 Tissue Kit)
Remove Sterivex filters from the –15 °C freezer.
Remove the Sterivex filter from the Whirl-Pak bag and remove both caps from the Sterivex cartridge.
Using a dedicated sterile 3 mL syringe for each filter, remove the storage buffer from the Sterivex cartridge. Attach the syringe to the inlet, introduce air into the syringe, and gently push to expel all liquid from the filter.
After discarding the liquid, close one end of the Sterivex cartridge with a luer cap.
Slowly pipette 720 µL Buffer ATL directly onto the filter through the inlet, avoiding backflow.
Slowly pipette 80 µL Proteinase K directly onto the filter.
Secure the Sterivex cartridge with the luer cap.
Shake by hand for several seconds to mix the reagents.
Place all filters on the roller shaker inside the incubator.
Incubate at 56 °C for ~16 hours, (minimum 4 hours) while rotating at approximately 400 rpm.
Day 2 – DNA Extraction
Clean the bench area and chairs first with 5% bleach, rinse with water to remove bleach residues, and then disinfect with 70% ethanol. Apply RNase Away to all equipment that will be used.
Soak all tube racks in 10% bleach solution, followed by three rinses with DI water. Allow racks to dry and expose to UV light for 15 minutes.
UV-sterilize LoBind tubes for 15 minutes in pre-sterilized tube racks and label with sample numbers:
- 1.5/2 mL LoBind tubes: (# samples + extraction blank) × 3
- 5 mL LoBind tubes: (# samples + extraction blank)
- Additional tubes to aliquot pre-warmed Buffer AE (Step 15)
Open and label additional materials from the Qiagen DNeasy Blood 26 Tissue Kit:
- DNeasy Mini spin columns: (# samples + extraction blank)
- Collection tubes: (# samples + extraction blank) × 2
Wipe down 1000 µL, 200 µL, 100 µL, and 10 µL pipettes with RNase Away and expose them to UV light for 15 minutes on each side.
Place the 50 mL tube of molecular-grade ethanol in a freezer (–20 °C) or on ice.
Heat an aliquot of Buffer AE (Qiagen DNeasy Blood 26 Tissue Kit) in a heating block at 70 °C.
Prepare the volume according to:
Volume = (n × 100 µL) + 15 µL
Prepare cryo-labels for DNA storage tubes (two of the three 1.5/2 mL LoBind tubes previously sterilized).
Assemble additional reagents and materials:
- Sterile 3 mL syringes (one per sample)
- Buffer AL
- Buffer AW1
- Buffer AW2
Remove Sterivex filters from the incubator.
Recover lysate from each filter.
Shake filters vigorously for several seconds.
Remove both caps from the Sterivex cartridge.
Attach a sterile 3 mL syringe to the inlet of the Sterivex cartridge and withdraw approximately 700 µL of lysate. Record the recovered volume and transfer it to a sterile 2 mL tube.
Prepare an extraction blank by adding 700 µL of ultrapure water to a sterile 2 mL tube (optional).
For each sample and extraction blank, add Buffer AL and 100% ethanol (pre-chilled) to the recovered lysate in a 1:1:1 ratio (lysate : AL : ethanol).
Vortex for 10 seconds to homogenize the mixture.
Load samples onto spin columns.
Pipette up to 650 µL of the mixture into a DNeasy Mini Spin column placed in a collection tube.
Centrifuge for 1 minute at 6000 × g (≈8000 rpm).
Discard the flow-through and return the spin column to the same collection tube.
Repeat Steps 22.1–22.3 until the entire sample has passed through the column.
Place the spin column in a new collection tube and add 500 µL Buffer AW1.
Centrifuge for 1 minute at 6000 × g (≈8000 rpm).
Discard the flow-through and collection tube.
Place the spin column in a new collection tube and add 500 µL Buffer AW2.
Centrifuge for 3 minutes at 20,000 × g (≈14,000 rpm) to dry the membrane.
Discard the flow-through and return the spin column to the same collection tube.
Place the spin column back into the collection tube and centrifuge again for 1 minute at 20,000 × g (≈14,000 rpm) to remove residual ethanol.
Transfer the spin column to a new sterile 1.5 mL microcentrifuge tube, pre-labeled with the sample ID.
Add 50 µL Buffer AE directly onto the membrane.
Incubate at room temperature (20–25 °C) for 1 minute.
Centrifuge for 1 minute at 8000 rpm.
Do not discard the eluate, as it contains the extracted DNA.
Repeat the elution by adding an additional 50 µL Buffer AE directly onto the membrane and centrifuge again for 1 minute at 8000 rpm.
Discard the spin column.
Store the eluted DNA at –20 °C.
Final DNA extract volume should be approximately 100 µL per sample.