May 27, 2026

A Modified Two-Stage Extraction Protocol for High-Purity eDNA Recovery from Aquatic Filter Membranes and Enclosed Capsule Filters

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvojxn7g4o/v1
  • Yang Fong1
  • 1CSU eDNA Laboratory, Gulbali Institute, Charles Sturt University, Albury-Wodonga NSW 2640, Australia
  • eDNA Laboratory Manual
Yang Fong
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvojxn7g4o/v1
Protocol Citation: Yang Fong 2026. A Modified Two-Stage Extraction Protocol for High-Purity eDNA Recovery from Aquatic Filter Membranes and Enclosed Capsule Filters. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvojxn7g4o/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: May 27, 2026
Last Modified: May 27, 2026
Protocol  Integer ID: 317988
Keywords: nucleic acid extraction, proteinase-k, guanidine hydrochloride, chaotropic agent, enzymatic lysis, biofilm-associated DNA, turbid water, Environmental DNA, Metabarcoding, Genomics, Biodiversity, purity edna recovery from aquatic filter membrane, enclosed capsule filters dna recovery, applicable to all common aquatic filter membrane format, common aquatic filter membrane format, aquatic filter membrane, capsule filter, column silica purification, desorption efficiency from the silica membrane, silica membrane, filter substrate, concentration chaotropic salt buffer, compounds prevalent in lowland freshwater system, biofilm glycoprotein, biofilm extracellular matrix, extracted nuclease, optimised lysis chemistry, purity edna recovery, ensuring complete enzymatic digestion, capsule sample, stage extraction protocol for high, biofilm, spectrum enzymatic digestion, integrity for quantitative pcr, chaotropic salt disruption
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Abstract
DNA recovery from aquatic filter membranes is the pre-amplification step most consequential to eDNA survey sensitivity. Residual particle matrices, biofilm glycoproteins, extracellular nucleoprotein complexes, and co-adsorbed environmental inhibitors particularly humic substances and polyphenolic compounds prevalent in lowland freshwater systems collectively undermine the performance of tissue-optimised lysis chemistries when applied to filter substrates. This protocol describes a purpose-developed, scalable extraction workflow that addresses these limitations through a sequential combination of chaotropic salt disruption and broad-spectrum enzymatic digestion, followed by spin-column silica purification. The method is applicable to all common aquatic filter membrane formats used in both active and passive sampling programmes, including nylon, mixed cellulose ester, cellulose nitrate, glass fibre, polycarbonate track-etched pads, cotton-based and Sterivex-GP polyethersulfone (PES) enclosed capsule filters.

The innovation of this protocol resides in four integrated design choices that together achieve performance beyond standard kit-based approaches on environmentally complex matrices: (i) a high-concentration chaotropic salt buffer at 4 M that simultaneously disrupts surface-adsorbed protein-DNA complexes, inactivates co-extracted nucleases, and conditions the lysate for silica binding without an intermediate pre-treatment step; (ii) a high proteinase K load of 2 mg mL⁻¹ throughout an extended overnight incubation, ensuring complete enzymatic digestion of structural proteins and biofilm extracellular matrix; (iii) a format-specific dual-pathway architecture that unifies open-pad and enclosed-capsule samples under a single downstream workflow from lysate clarification onward; and (iv) a warm dual-fraction elution strategy that maximises desorption efficiency from the silica membrane and recovers both membrane-adsorbed and solution-phase DNA populations in a single pooled eluate. The resulting DNA is of sufficient purity and integrity for quantitative PCR and next-generation sequencing library preparation targeting vertebrate 12S rRNA, invertebrate COI, and broader multi-kingdom metabarcoding marker systems.
Guidelines
Filter membrane material compatibility. The lysis chemistry and procedural parameters have been developed and validated for nylon, mixed cellulose ester, cellulose nitrate, glass fibre, and Sterivex-GP polyethersulfone membrane formats. Positively charged nylon membranes (e.g., Biodyne C or B) retain DNA through electrostatic interactions in addition to physical filtration; a brief pre-wash with 200 μL UltraPure water through the sectioned membrane prior to lysis buffer addition is recommended to release electrostatically bound DNA. Polycarbonate track-etched membranes are physically rigid and do not dissolve under the lysis conditions described; their compatibility should be verified empirically for each supplier lot before large-scale batch use.

Lysis stringency scaling. The proteinase K concentration of 2 mg mL^^−1 and chaotropic salt concentration of 4 M are calibrated to the inhibitor loads characteristic of turbid lowland floodplain rivers within the Murray-Darling Basin. For oligotrophic, marine, or low-turbidity samples where particle and inhibitor loads are substantially lower, the proteinase K concentration may be reduced to 1 mg mL^^−1 without measurable yield loss, with a corresponding reduction in reagent cost per sample.

Applicability to sedimentary ancient DNA. Eluates produced by this protocol are compatible with downstream workflows for degraded and sedimentary ancient DNA (sedaDNA), including short-read amplicon approaches and hybridisation capture library preparation. A fragment size distribution assessment using a Bioanalyzer HS DNA chip is recommended before amplicon library preparation with primers targeting amplicons shorter than 150 bp.

Optional mechanical lysis pre-treatment. Bead-beating mechanical disruption is not incorporated as a default step, to preserve fragment length for library preparation applications. For samples from environments where cells with recalcitrant walls are expected to predominate (e.g., cyanobacterial or chlorophyte-dominated water bodies), a brief bead-beating step (30 s at 5-6 m s^^−1 with 0.5 mm zirconia-silica beads) prior to lysis buffer addition may improve DNA liberation from intact cells. Users should evaluate this modification empirically against their library fragment length requirements.
Materials
Lysis Master Buffer - Prepare Fresh; 1.0 mL per Sample

  1. Guanidinium hydrochloride, 5 M (in 10 mM Tris-HCl pH 8.0): 800 µL, Final Concentration: 4.0 M
  2. EDTA disodium, 0.5 M, pH 8.0: 50 µL, Final Concentration: 25 mM
  3. SDS, 20% w/v aqueous solution: 25 µL, Final Concentration: 0.5% w/v
  4. Proteinase K, 20 mg mL⁻¹: 100 µL, Final Concentration: 2.0 mg mL⁻¹
  5. UltraPure DNase/RNase-free water: 25 µL

Total: 1,000 µL

Materials

  • Guanidinium hydrochloride (Sigma-Aldrich G3272; ≥98.0% purity)
  • Proteinase K, 20 mg mL⁻¹ aqueous solution (Qiagen Cat. 19133; or Thermo Fisher E00491)
  • EDTA disodium, 0.5 M, pH 8.0 (Ambion AM9261; molecular biology grade)
  • SDS solution, 20% w/v (Sigma-Aldrich 05030; molecular biology grade)
  • Qiagen DNeasy Blood 26 Tissue Kit (Qiagen Cat. 69506 (50-prep) or 69504 (250-prep))
  • Absolute ethanol, molecular biology (Merck or equivalent; ≥99.9% v/v)
  • Tris-HCl, 10 mM, pH 8.0 (Ambion AM9855G; molecular biology grade)
  • Buffer AE (DNeasy Kit component) (Qiagen; 10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0)
  • UltraPure DNase/RNase-free water (Invitrogen Cat. 10977015 or equivalent)
  • Sodium hypochlorite solution, 10% v/v (Freshly prepared from commercial 12.5% stock)
  • Ethanol, 70% v/v (Prepared from absolute ethanol in UltraPure water)
  • Longmire's buffer (Sterivex only; In-house: 100 mM Tris pH 8.0, 100 mM EDTA, 10 mM NaCl, 0.5% SDS)

Consumables

  • Eppendorf DNA LoBind tubes, 2.0 mL (Cat. 0030108078) - reduced-binding surface; use throughout extraction
  • Eppendorf DNA LoBind tubes, 1.5 mL (Cat. 0030108051) - for eluates, aliquots, and archival storage
  • LoBind DNA tubes, 5.0 mL (Eppendorf Cat. 0030122348) - for lysate pooling and AL-conditioning step
  • Sterile scalpel blades, size 22 - single-use disposable; one new blade per filter pad
  • Sterile forceps, two pairs per session - autoclaved; UV-treated inside cabinet immediately prior to use
  • Sterile Petri dishes, 90 mm - single-use disposable; UV-treated prior to filter manipulation
  • Aerosol-barrier filter tips, 10-200 µL - RNase/DNase-free certified; mandatory for all pre-amplification steps
  • Aerosol-barrier filter tips, 200-1000 µL - RNase/DNase-free certified; mandatory for all pre-amplification steps
  • Powder-free nitrile examination gloves - change between every individual sample
  • Sterile luer-lock syringes, 5 mL - Sterivex Pathway S only
  • Sterivex inlet and outlet caps (MoBio/Qiagen) - Pathway S only; for capsule sealing during lysis incubation
  • QIAShredder spin columns (Qiagen Cat. 79656) - glass fibre membrane format only, at Step 9
  • Qubit Assay Tubes (Thermo Fisher Cat. Q32856) - fluorometric DNA quantification
  • Parafilm M - Sterivex capsule end-sealing during lysis incubation (Pathway S only)

Equipment

  • Class II laminar flow cabinet with UV germicidal lamp - UV-treat interior surfaces for ≥30 min before and after each session
  • ThermoMixer C (Eppendorf) or equivalent - temperature accuracy ±0.5 °C; programmable to 56 °C at 300-850 rpm
  • Refrigerated microcentrifuge - rated to ≥20,000 x g; Eppendorf 5427R or equivalent
  • Vortex mixer - variable speed; Vortex-Genie 2 or equivalent
  • Rotating end-over-end mixer - 15-20 rpm; Stuart SB3 or equivalent (Sterivex Pathway S)
  • Qubit 3 or 4 Fluorometer - with dsDNA High Sensitivity Assay Kit
  • Nanodrop 2000 Spectrophotometer - for A260/A280 and A260/A230 purity ratio assessment
  • UV crosslinker or transilluminator (254 nm) - for decontamination of consumables and tube racks
  • Dedicated pre-PCR laboratory space - physically isolated from post-amplification work areas; positive-pressure ventilation preferred
Safety warnings
HAZARDS WARNING - GUANIDINIUM SALTS AND BLEACH: INCOMPATIBILITY

Guanidinium salts react with sodium hypochlorite (bleach) to generate cyanogen chloride, a toxic gas with acute respiratory hazard. These two reagents must never be combined directly, and their waste streams must be collected and disposed of separately. All laboratory surfaces and equipment contaminated with guanidinium-containing buffers must be decontaminated with 70% ethanol only. Bleach-based surface decontamination may be applied only after complete removal of guanidinium residue through prior ethanol treatment. All guanidinium-containing waste must be collected in clearly labelled chemical waste containers and disposed of according to institutional chemical waste procedures.

Guanidinium hydrochloride: classified as an eye, skin, and respiratory irritant (GHS07). Prepare and handle all guanidinium-containing solutions within a well-ventilated Class II laminar flow cabinet. Wear chemical-resistant nitrile gloves (minimum 4 mil), a full-length laboratory coat, and splash-rated safety glasses. In the event of ocular or dermal contact, irrigate with copious water for a minimum of 15 minutes and seek medical advice.

Proteinase K: classified as a respiratory and dermal sensitizer with repeated occupational exposure (GHS08). Use only ready-prepared liquid solution (20 mg mL⁻¹) to avoid aerosolization. Aerosol-barrier filter tips are mandatory for all proteinase K pipetting operations.

Sodium dodecyl sulphate (SDS): irritant and flammable at elevated temperature. Avoid generating aerosols during transfer; confirm full dissolution before incorporating into the lysis master buffer.
Ethics statement
No human participants, human biological material, or regulated animals were involved in the development or validation of this protocol. Fieldwork was conducted under appropriate site access permissions and license.
Before start
This protocol accommodates all aquatic filter membrane formats in routine use across active and passive eDNA sampling methodologies. Format-specific procedural modifications are incorporated within the relevant protocol steps.

  • The GuHCl-SDS-EDTA base buffer (without proteinase K) may be prepared in larger batches and stored at room temperature for up to 7 days. Add proteinase K only on the day of extraction.
  • Scale master mix volume to: (n samples + 1 extraction blank) x 1.0 mL, with 10% preparation overage.
Procedure
PATHWAY NOTE: Steps 1-3 (Phase 1) apply to all filter formats. At Phase 2, follow the appropriate pathway: Pathway A (Steps 4-6) for all open membrane filter pads, or Pathway S (Steps 4S-6S) for Sterivex-GP enclosed capsule filters. Both pathways merge at Step 7 (Phase 3) and proceed identically through Step 16.
Phase 1; Laboratory Preparation and Contamination Control
Step 1. Laminar Flow Cabinet Preparation and Decontamination
Activate the laminar flow cabinet UV lamp and irradiate the interior work surface for a minimum of 30 minutes prior to commencing any work. During this period, decontaminate all external work surfaces, micropipette bodies, tube racks, forceps handles, and ThermoMixer heat-block surfaces with 10% sodium hypochlorite solution (2 min contact time), followed by 70% ethanol. Allow all surfaces to dry completely before introducing any sample material. Confirm that the ThermoMixer or water bath has reached 56 °C (±0.5 °C) and is stable. Prepare two pre-warming aliquots of Buffer AE (100 µL each) per sample in sealed 1.5 mL LoBind tubes and place them in the 56 °C block for use at Steps 14 and 15.
Timing: minimum 30 min UV irradiation; ThermoMixer must be stable at 56 °C before extraction begins.
Step 2. Preparation of Lysis Master Buffer
Prepare the lysis master buffer as described in the Reagent Preparation section. Calculate the required volume as: (n samples + 1 extraction blank) x 1.0 mL, with 10% overage. Combine components in the order specified, adding proteinase K as the final component immediately before dispensing. Homogenise by brief vortexing (3 s). Hold on ice until required.
Step 3. Negative Extraction Blank Preparation
Prepare one negative extraction blank (EB) per batch of up to 12 samples. For open membrane formats: dispense 100 µL of UltraPure water into a pre-labelled 2 mL LoBind tube. For Sterivex format: dispense 200 µL of UltraPure water. The EB must be carried through every subsequent step; including all incubations, washes, and elution steps, using identical volumes and conditions to field samples. A positive EB signal in downstream qPCR or amplicon sequencing constitutes evidence of extraction-stage contamination. All samples from the affected batch must be discarded, and full decontamination must be completed before re-extraction.
IMPORTANT: Do not proceed with amplification from any batch in which the extraction blank exceeds 0.05 ng µL⁻¹ on fluorometric quantification.
Phase 2A; Open Membrane Filter Pad Preparation (Pathway A)
Apply Steps 4-6 to nylon, MCE, cellulose nitrate, and polycarbonate track-etched filter pads. For glass fibre membranes, apply the glass fibre modification within Step 5 and proceed to Step 6.
Step 4. Membrane Recovery and Inspection
Don fresh nitrile gloves. Using autoclaved forceps, retrieve the filter membrane from its storage vessel (2 mL tube, sealed cryovial, or equivalent). Transfer the membrane to a sterile UV-treated 90 mm Petri dish within the laminar flow cabinet. If the membrane was stored in aqueous preservation buffer (Longmire's solution, 100% ethanol, or LifeGuard), drain excess preservative by tilting the membrane with forceps and blotting the outer edge against a sterile Kimwipe. Do not allow the membrane surface to desiccate. Record all observations relevant to extraction quality in the laboratory notebook: degree of particulate loading, visible biofilm, membrane colouration, structural integrity, and any departure from normal appearance.

IMPORTANT: Gloves must be changed between each individual sample without exception.
Step 5. Membrane Sectioning
For nylon, MCE, cellulose nitrate, and PCTE membranes: using a fresh sterile scalpel blade, section the membrane into 8-12 strips of 1-2 mm width while it remains within the Petri dish. Sectioning increases the effective surface-area-to-volume ratio during lysis incubation and physically disrupts compressed surface particulate material. Transfer all membrane sections and any particulate debris to a pre-labelled 2 mL LoBind tube using autoclaved forceps. Add 100 µL UltraPure water to the Petri dish, swirl to recover residual material, and transfer this wash to the same tube. Sediment briefly at 100 x g for 5 s. Glass fibre membranes only: do not section. Transfer the intact membrane whole to a 2 mL LoBind tube. A QIAshredder spin column will be required at Step 9 to prevent fibre fragments from obstructing the DNeasy spin column during lysate loading.
Step 6. Lysis Buffer Addition and Overnight Incubation (Pathway A)
Pipette 900 µL of lysis master buffer directly onto the membrane material in the 2 mL LoBind tube, ensuring complete submersion of all fragments. If any fragments protrude above the buffer surface, gently depress them using a clean barrier-tip pipette. Close the tube securely and vortex at maximum speed for 15 s. Transfer all tubes to the ThermoMixer at 56 °C, 850 rpm, for a minimum of 14 h; 16 h is preferred for heavily loaded membranes. If a ThermoMixer is unavailable, use a rotating end-over-end mixer (15-20 rpm) inside a 56 °C incubator over the same duration. After incubation, proceed to Step 7 (Phase 3).
Phase 2S; Sterivex-GP Capsule Preparation (Pathway S)
Apply Steps 4S-6S to Sterivex-GP 0.22 µm polyethersulfone enclosed capsule filters. Two fractions are extracted and processed in parallel: SXCAPSULE (capsule interior lysate) and SXTUBE (expelled preservation buffer pellet). Both fractions are loaded sequentially onto the same DNeasy spin column at Step 9.
Step 4S. Sterivex Capsule Surface Decontamination and Inspection
Wipe the external surface of the capsule with 70% ethanol and allow 60 s contact time. Inspect the inlet and outlet caps and parafilm seals for physical integrity. Note any seal compromise in the extraction record, as such samples carry elevated risk of external contamination or accelerated DNA degradation. Change gloves between each individual capsule.
Step 5S. Preservation Buffer Evacuation and Pellet Preparation (SXTUBE)
Remove the inlet cap. Attach a sterile 5 mL luer-lock syringe to the inlet port and evacuate all preservation buffer from the capsule interior by gentle aspiration. Transfer the recovered buffer to a pre-labelled 2 mL LoBind tube (SXTUBE). Record the recovered volume. Replace the inlet cap on the capsule. Centrifuge the SXTUBE fraction at 6,000 x g for 30 min at room temperature to pellet particulate material. Carefully decant the supernatant without disturbing the pellet. If no visible pellet is apparent, retain the terminal 20 µL of supernatant. Add 180 µL of lysis master buffer to the SXTUBE pellet and hold on ice.
Step 6S. Lysis Buffer Introduction and Overnight Incubation (SXCAPSULE)
Through the inlet port of the Sterivex capsule, introduce 720 µL of lysis master buffer using a filter-tip pipette. Close the inlet cap and confirm the outlet cap is secure. Seal both ends with parafilm. Invert the sealed capsule vigorously 10 times to distribute lysis buffer across all internal membrane surfaces. Place the sealed SXCAPSULE on a rotating end-over-end mixer at room temperature for 10 min to allow buffer equilibration, then transfer to a 56 °C incubator on the rotator. Transfer the SXTUBE tube to the ThermoMixer at 56 °C, 850 rpm, simultaneously. Incubate both fractions overnight for 14-16 h. After incubation, process SXCAPSULE and SXTUBE separately through Steps 7-9, then load both fractions sequentially onto the same spin column at Step 9.
Phase 3; Lysate Clarification and Column Conditioning (All Filters Type)
Step 7. Post-Incubation Homogenisation and Clarification Centrifugation
Remove all tubes from the ThermoMixer and allow to equilibrate to room temperature for 5 min. Vortex each tube at maximum speed for 20 s.

  • Open membrane pads: centrifuge at 6,000 x g for 5 min at room temperature. Transfer the clarified supernatant to a pre-labelled 5 mL LoBind tube; record the recovered volume. Discard the membrane pellet.
  • Glass fibre membranes: pass the post-vortex lysate through a QIAShredder spin column in a 2 mL collection tube; centrifuge at 14,000 x g for 2 min; transfer the filtrate to a 5 mL LoBind tube.
  • Sterivex SXCAPSULE: remove the inlet cap and aspirate all capsule interior lysate into a 5 mL LoBind tube using a 5 mL luer-lock syringe.
  • Sterivex SXTUBE: vortex 15 s, then centrifuge at 6,000 x g for 2 min; transfer the cleared supernatant to a separate 5 mL LoBind tube.

Step 8. Lysate Conditioning for Silica Binding
To each 5 mL LoBind tube containing clarified lysate, add Buffer AL in a volume equal to the recovered lysate, followed by an equal volume of ice-cold 100% ethanol. Maintain the volumetric ratio throughout at lysate : Buffer AL: ethanol = 1:1:1. Homogenize by vortexing at maximum speed for 20 s.

CRITICAL: This ratio must be maintained for optimal DNA adsorption to the silica membrane. If the total conditioned volume exceeds 1.8 mL, load excess onto the same spin column in sequential aliquots at Step 9; do not use additional columns for the same sample.

For Sterivex: condition SXCAPSULE and SXTUBE fractions independently; both are loaded onto a single spin column at Step 9.
Phase 4; Silica Spin-Column Purification
Step 9. Sequential Loading of Conditioned Lysate onto DNeasy Spin Column
Insert a DNeasy Mini Spin Column into a 2 mL collection tube. Apply 650 µL of conditioned lysate to the membrane. Centrifuge at 6,000 x g for 1 min at room temperature. Discard the flow-through and collection tube; transfer the spin column to a fresh 2 mL collection tube. Repeat for successive 650 µL aliquots until all conditioned lysate has been applied.

For Sterivex samples: load all SXCAPSULE aliquots first, then load all SXTUBE aliquots onto the same column.

Step 10. First Wash - Buffer AW1
Transfer the spin column to a new 2 mL collection tube. Apply 500 µL Buffer AW1 to the membrane. Centrifuge at 6,000 x g for 1 min at room temperature. Discard the flow-through and collection tube.

Step 11. Second Wash - Buffer AW2 with Mandatory Desiccation Spin
Transfer the spin column to a new 2 mL collection tube. Apply 500 µL Buffer AW2 to the membrane. Centrifuge at 20,000 x g for 3 min at room temperature. Discard the flow-through; retain the collection tube in place. Without replacing the collection tube, perform a second centrifugation at 20,000 x g for 1 min (desiccation spin). This mandatory step ensures complete ethanol removal from the silica membrane; residual ethanol carryover to the eluate is a frequent and preventable cause of downstream PCR inhibition. Transfer the spin column to a fresh 2 mL collection tube. Allow the open column to stand at room temperature for 3 min to permit passive evaporation of trace ethanol.
Phase 5; Warm Dual-Fraction Elution
Step 12. Primary Elution
Transfer the desiccated spin column to a 1.5 mL DNA LoBind tube from which the cap has been removed. Apply 60 µL of pre-warmed Buffer AE (56 °C) to the centre of the silica membrane; ensure complete coverage without direct contact between the pipette tip and membrane surface. Incubate the column within the LoBind tube at 56 °C for 10 min with gentle agitation at 300 rpm or by periodic manual inversion. Centrifuge at 6,000 x g for 1 min at room temperature. The primary eluate is retained in the LoBind tube.

Step 13. Secondary Elution and Eluate Pooling
Return the spin column to the same LoBind tube containing the primary eluate. Apply 40 µL of pre-warmed Buffer AE (56 °C) to the membrane centre. Incubate at 56 °C for 5 min with gentle inversion (5-6 times). Centrifuge at 6,000 x g for 1 min at room temperature. Discard the spent spin column. The pooled primary and secondary eluates comprise the final DNA extract, with a combined volume of approximately 90-100 µL.
Phase 6; Quality Assessment and Sample Archiving
Step 14. Fluorometric DNA Quantification
Quantify total dsDNA concentration in each eluate using the Qubit dsDNA High Sensitivity Assay Kit, following the manufacturer's instructions with 2 µL of eluate per reaction. Record ng µL⁻¹ values for all samples and the extraction blank. Expected yield range: 0.5-20 ng µL⁻¹, dependent on filter type, sampling duration, and water body turbidity. If the extraction blank exceeds 0.05 ng µL⁻¹, the extraction batch is failed. Do not proceed with amplification. Investigate the contamination source, repeat decontamination, replace all reagent aliquots, and re-extract.

Step 15. Spectrophotometric Purity Assessment
Assess DNA purity using 2 µL of eluate on the Nanodrop 2000. Record A260/A280 and A260/A230 ratios for all samples.

Target purity thresholds for downstream NGS applications:

  • A260/A280:  1.70-2.00 (below 1.70 indicates residual protein co-extraction; above 2.00 suggests RNA contamination or single-stranded nucleic acid predominance)
  • A260/A230: 1.50-2.20 (below 1.50 indicates carryover of chaotropic salt, humic substances, polysaccharides, or residual organic solvent)

For samples with A260/A230 below 1.30, apply post-extraction inhibitor removal using the Qiagen PowerClean Pro DNA Clean-Up Kit before amplification. Confirm amplifiability of cleaned samples using an internal positive control (IPC) qPCR assay.

Step 16. Aliquoting and Archival Storage
Immediately following quality assessment, partition each eluate into a minimum of three single-use aliquots in 1.5 mL LoBind tubes. Label all aliquots with sample identifier, extraction date, aliquot designation, and measured concentration.

  • Aliquot A;  Working stock: 20-30 µL; store at -20 °C for routine qPCR and library preparation. Thaw on ice; limit to a single freeze-thaw cycle per aliquot.
  • Aliquot B;  Long-term archive: 30-40 µL; store at -80 °C as permanent laboratory record.
  • Aliquot C;  Inhibition control reserve: 10-15 µL; store at -20 °C; reserved exclusively for IPC assay validation before quantitative applications.

Extracted eDNA should not be stored at 4 °C for periods exceeding 72 h. Iterative freeze-thaw cycling causes progressive DNA fragmentation and is incompatible with fragment-size-sensitive downstream applications.
Troubleshooting
Troubleshooting guide for common observations encountered during eDNA extraction using this protocol


Expected Results
Performance parameters are based on internal trial-and-error testing conducted at the CSU eDNA Laboratory using filter membranes collected from various temperate lowland freshwater systems. Actual values vary depending on water body type, sampling duration, season, and filter format.
ABC
ParameterObserved RangeNotes
Total dsDNA yield (Qubit HS) 0.5-20 ng µL⁻¹ in 90-100 µL eluate Higher in turbid, particle-rich systems; lower in oligotrophic or short duration samples
A260/A280 absorbance ratio 1.75-1.98 Acceptable for qPCR, and NGS library preparation without further clean-up
A260/A230 absorbance ratio 1.60-2.10 Values below 1.50 require dilution or PowerClean Pro clean-up prior to amplification
Extraction blank (Qubit HS) Below detection or ≤0.02 ng µL⁻¹ Batch pass threshold set at 0.05 ng µL⁻¹; values above require batch rejection
Sterivex SXCAPSULE + SXTUBE (pooled) 10-30% higher than SXCAPSULE alone Dual-fraction pooling recovers significant DNA concentrated in expelled preservation buffer; recommended as standard practice
Protocol references
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5. Milián-García, Y. (2022). Modified Qiagen DNeasy Blood and Tissue extraction method for eDNA extraction from filters. protocols.io. https://doi.org/10.17504/protocols.io.8epv59y4jg1b/v1

6. Pawlowski, J., et al. (2022). Environmental DNA metabarcoding for benthic monitoring: A review of sediment sampling and DNA extraction methods. Science of the Total Environment, *818*, Article 151783. https://doi.org/10.1016/j.scitotenv.2021.151783

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8. Bessey, C., et al. (2021). Passive eDNA collection enhances aquatic biodiversity analysis. Communications Biology, *4*, Article 236. https://doi.org/10.1038/s42003-021-01760-8

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Acknowledgements
The author pays respect to Wiradjuri Elders past and present and acknowledges the enduring ecological knowledge embedded in Country that continues to motivate this research. The author also acknowledges the support of the Gulbali Institute and Charles Sturt University, Albury-Wodonga. The author also declares no competing financial or non-financial interests in relation to this protocol.