Jun 28, 2026

OceanOmics Centre: eDNA Extraction from Filter Papers with modified Promega Maxwell® PureWater Kit

DOI
https://dx.doi.org/10.17504/protocols.io.36wgqpjwxvk5/v1
OceanOmics Centre: eDNA Extraction from Filter Papers with modified Promega Maxwell® PureWater Kit
  • Laura Missen1,
  • Anna Depiazzi1,
  • Marcelle Ayad2,
  • Ebony Thorpe1,
  • OceanOmics UWA1
  • 1Minderoo OceanOmics Centre at UWA;
  • 2Minderoo Foundation
Laura Missen
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgqpjwxvk5/v1
External link: https://www.uwa.edu.au/oceans-institute/partnerships/minderoo-oceanomics-centre-at-uwa
Protocol Citation: Laura Missen, Anna Depiazzi, Marcelle Ayad, Ebony Thorpe, OceanOmics UWA 2026. OceanOmics Centre: eDNA Extraction from Filter Papers with modified Promega Maxwell® PureWater Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqpjwxvk5/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: August 12, 2025
Last Modified: June 28, 2026
Protocol  Integer ID: 224590
Keywords: environmental DNA, eDNA, DNA extraction, DNA extraction from filter paper, Promega Maxwell, nucleic acid extraction, marine eDNA, Buffer ATL, CTAB, Proteinase K digestion, Illumina Sequencing , Metabarcoding, oceanomics centre, rsc purewater kit, rsc purewater kit for use, dna extracts for metabarcoding, automated nucleic acid purification platform, nucleic acid purification platform, dna extract, mixed cellulose ester filter membrane, digestion to extraction, modified extraction, extraction from filter paper, extraction, generation sequencing, yielding dna
Abstract
This protocol describes a modified extraction workflow that adapts the Promega Maxwell® RSC PureWater Kit for use with the Maxwell® RSC 48 automated nucleic acid purification platform. The protocol was optimised for 47 mm mixed cellulose ester filter membranes, preserved in Buffer ATL (Qiagen). It enables simultaneous processing of up to 48 samples, yielding DNA suitable for high-throughput amplicon sequencing on Illumina platforms. From digestion to extraction, 48 samples will take approximately 5-6 hours.


This method is employed by the OceanOmics Centre to generate DNA extracts for metabarcoding and Next-Generation Sequencing. It represents a compiled and modified protocol based on Promega's published methods, adapted for our specific sample type. This protocol has been validated with both the Maxwell® RSC PureWater Kit and the Maxwell® RSC PureFood GMO and Authentication Kit.
Guidelines
This protocol is suitable for DNA extraction from 47 mm mixed cellulose membrane filters. The filters should be stored in 2.0 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 with 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 (including suitable 0.5 mL tube adapters if required)
  • Incubator shaker
  • UV sterilisation cabinet
Equipment
Promega Maxwell RSC 48
NAME
Automated Nucleic Acid Extraction
TYPE
Promega
BRAND
AS8500
SKU
https://www.promega.com.au/products/lab-automation/automated-dna-rna-extraction-purification-maxwell/maxwell-rsc-48-instrument/?tabset0=0
LINK
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
Buffer ATLQiagenCatalog #19076 OR CTABPromega CorparationCatalog #MC1411
Maxwell RSC PureWater KitPromega CorparationCatalog #AS2110 or Maxwell RSC PureFood GMO and Authentication KitPromega CorparationCatalog #AS1600
Proteinase KPromega CorparationCatalog #MC111
Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32851



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 a lab coat, gloves, and safety glasses when handling kit components.
  • Dispose of all waste following local hazardous waste disposal.
Chemical Hazards:
  • Buffer ATL and Lysis Buffer contain Guanidine Hydrochloride. This is harmful if swallowed, inhaled, or in contact with skin.
  • Reagent cartridges contain 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 and cartridges are stored as recommended in manufacturer's protocol.
  • Conduct in a well-ventilated area or fume hood if available.
  • Do not reuse cartridges, they are single use only.
Link to SDS documents: Download Maxwell RSC PureFood and GMO and Authentication Kit SDS.pdfMaxwell RSC PureFood and GMO and Authentication Kit SDS.pdf196.2KB Download Buffer ATL SDS.pdfBuffer ATL SDS.pdf215.9KB Download CTAB SDS.pdfCTAB SDS.pdf225.9KB Download Maxwell RSC PureWater SDS.pdfMaxwell RSC PureWater SDS.pdf1.2MB

Before start
  1. Pre-heat incubator shaker to 56 °C .
  2. Aliquot the required volume of reagents (ATL, ProK, Lysis buffer, Elution buffer).
  3. Sterilise scissors and tweezers (refer to Guidelines).
  4. Thaw samples at room temperature for 30 minutes.
  5. Wipe the Promega Maxwell® 48 trays with 70% ethanol, then conduct a sterilisation run for 15 minutes.
  6. Label and UV sterilise tubes:
  • One 2.0 mL DNA LoBind tube per sample for transferring half the filter paper to.
  • Two 1.5 mL DNA LoBind tubes per sample for DNA extracts (one for an archive stock and one for a working stock).
  • One 0.5 mL tube per sample for collecting the eluate in the Promega Maxwell.


Digestion
(Optional) To maximise yield and volume from each sample, filter papers can be cut in half, extracted separately, and then combined once eluted.

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 tube. Sterilise equipment as per the guidelines.
Add 540 µL Buffer ATL to all the secondary tubes created in step 1.

Using sterilised scissors, and keeping the filter membrane in the tube, cut the filter paper into small pieces (<1 cm2) to increase exposed surface area.
After each implement comes into contact with the sample, sterilise using 10% bleach then rinse with ddH2O.
Add 60 µL Proteinase K to each sample. Change tip between each sample and pipette directly onto the filter membrane.
Vortex each sample for 15 seconds and spin-down briefly.

NOTE: ensure the whole filter paper is submerged.
Place tubes in a rack and place into the incubator shaker for 3 hours at 300 rpm, 56°C .

Once digestion is complete, samples can either be stored at -20°C, or proceed immediately to extraction.

This is a safe stopping point.
DNA can be stored at -20°C long-term. Avoid freeze/thaw cycles since this can degrade DNA.
Prepare the Maxwell® Cartridge
Turn on the Maxwell® instrument and Tablet PC. Ensure the tablet charger is inserted into the device.

NOTE: prior to use, wipe the deck trays with ethanol and perform UV light sterilisation on the Maxwell® for 15 minutes.
Open the door and remove the deck trays from the Maxwell®.
Place the number of required cartridges in the deck tray(s); with one cartridge required per 2.0 mL sample tube and one cartridge for the extraction blank. Place each cartridge in the deck tray with the #1 tube position facing away from the elution tube. Snap cartridge into position by firmly pressing down.
Cartridge image: Promega Maxwell® documentation

Carefully peel back the seals on the top of the cartridge.
Place a plunger into well #8 of each cartridge (closest to the elution tube position).

NOTE: use clean gloves when handling the plungers.
Add 300 µL of Lysis Buffer to well #1.

Label elution tubes (0.5 mL) then place into the elution position for each cartridge in the deck tray(s). Ensure tube lid remains open and faces towards the front of the instrument.

NOTE: use clean gloves when handling elution tubes.
Add 100 µL of Elution Buffer to the base of each elution tube.
Add up to 600 µL of lysate to well #1. Use the pipette tip to squeeze the filter paper to the side of the tube to obtain as much liquid as possible. Do this as many times as necessary and change tip between each sample.
To the extraction blank, add 540 µL buffer ATL and 60 µL Proteinase K to well #1.
Extraction
Start the Maxwell® software by double-touching the icon on the desktop.
Proceed through self-check and home all moving parts.
Touch Start to begin the process of running a method.
Select the PureWater method then press Proceed.

NOTE: or select the PureFood GMO and Authentication method if using this kit.
On the ‘Cartridge Setup’ screen, touch the cartridge positions to select/deselect the positions to be used for the run. Touch Proceed, the door will open.
Insert the deck trays. Hold the tray by the sides and ensure the trays are placed with the elution tubes closest to the door. Angle the back of the deck tray downward and place into the instrument so that the back of the deck tray is against the back of the instrument platform. Press down on the front of the tray firmly to lock the tray into the instrument platform.
Confirm that all 'Extraction Checklist' items have been performed.
  • Cartridges are loaded on the instrument.
  • Lysate is added to well #1.
  • Uncapped elution tubes are present with 100 µL of elution buffer.
  • Plungers are present in well #8.
Press Start to begin the run. The platform will retract, and the door will close.
At the end of the run, follow the on-screen instructions to open the door. Ensure all the plungers are in well #8 and not on the plunger bars.
Remove the deck tray(s) from the instrument.
Close the elution tubes and spin-down using 0.5 mL rotor adapters in the centrifuge. 8000 x g

Transfer the eluted DNA extracts into labelled Eppendorf 1.5 mL DNA LoBind tubes.

NOTE: due to evaporation in the heated elution, the total volume of eluate may fall below 100 µL.

If no duplicates exist per sample, pipette mix, then transfer 50 µL of eluted DNA into the pre-labelled 1.5 mL DNA LoBind tube for archiving.
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). b. Using a 200 µL pipette, pipette mix 10 times. c. Transfer 100 µL from tube B back to tube A.

This is a safe stopping point.
The working stock should be stored at 4°C until processed. Once processed store at -20°C. Archive stock should be stored at at -80°C.
Remove the cartridges and plungers from the deck tray.
Decant liquid from the cartridge into a labelled extraction waste bottle, discard plasticware.
Wipe the deck trays with 70% ethanol, place back into the instrument and close the door.

NOTE: do not use bleach or any corrosive substance on the Maxwell®.
Perform UV light sterilisation on the Maxwell® for 15 minutes.
DNA Quantification
Quantify 2 µL of each sample using Qubit HS dsDNA kit. Once quantified, samples are then plated for qPCR and Next-Generation Sequencing.
Protocol references