May 08, 2025
Dynabeads Wastewater Virus Enrichment & Total Nucleic Acid Extraction
- Victor Mabasa1,
- Natasha Singh1,
- Emmanuel Phalane1,
- Mokgaetji Macheke1,
- Thabo Mangena1,
- Lethabo Monametsi1,
- Lebohang Rabotapi1,
- Sipho Gwala1,2,
- Nkosenhle Ndlovu1,
- Fiona Els1,3,
- Sibonginkosi Maposa1,
- Said Rachida1,
- Kerrigan McCarthy1,2,
- Mukhlid Yousif1,2
- 1National Institute for Communicable Diseases;
- 2University of Witwatersrand;
- 3Gauteng City-Region Observatory
- Wastewater Genomics Syndicate
Protocol Citation: Victor Mabasa, Natasha Singh, Emmanuel Phalane, Mokgaetji Macheke, Thabo Mangena, Lethabo Monametsi, Lebohang Rabotapi, Sipho Gwala, Nkosenhle Ndlovu, Fiona Els, Sibonginkosi Maposa, Said Rachida, Kerrigan McCarthy, Mukhlid Yousif 2025. Dynabeads Wastewater Virus Enrichment & Total Nucleic Acid Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj9j5nlk5/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 28, 2025
Last Modified: May 08, 2025
Protocol Integer ID: 119215
Keywords: Wastewater, enrichment, dynabeads , virus enrichment
Disclaimer
This protocol was amended from Pub. No. MAN0025695 by appliedbiosystems
Abstract
The Applied Biosystems MagMAX Wastewater Ultra Nucleic Acid Isolation Kit (Cat. No. A52610) is specifically designed for virus enrichment and total nucleic acid extraction from 10 mL wastewater samples. The purified nucleic acids suit various downstream applications, including real-time PCR, digital PCR, and next-generation sequencing. Both the enrichment and extraction processes are automated on the KingFisher Flex Purification System with a 24-deep-well head, using in-house adapted programmes from MagMAX_Wastewater_10mL_Flex24.
Guidelines
General
- Perform all steps at room temperature (20–30°C), unless otherwise noted.
- Clean the work surfaces with RNaseZap to remove nucleases, then wipe the surfaces with 70% to 100% molecular biology grade ethanol to remove additional contaminants.
- Precipitates can form in the Lysis Buffer, Binding Solution, and Wash Buffer if stored below 20°C. If this occurs, warm the reagents at 37°C, then gently mix to dissolve the precipitates. Avoid creating bubbles.
Binding Bead Mix
- Vortex Binding Beads thoroughly before each use.
- Ensure that the beads stay fully mixed within the solution during pipetting.
- Avoid creating bubbles during mixing and aliquoting.
- The binding Bead Mix is very dense so pipet carefully to ensure that the correct volume is added to the sample
Materials
Reagents
Nuclease-free water
Ethanol, 100% (molecular biology grade)
MagMAX Wastewater Ultra Nucleic Acid Isolation Kit (A52610)
Consumables
RNase-Free Microfuge Tubes, 1.5 mL
KingFisher Flex 24 Deep-Well Plate
MicroAmp Clear Adhesive Film
Conical Tubes (50 mL)
Equipment
KingFisher Flex Purification System with 24 deep‑well head
Standard laboratory vortex
Pipettes
Automation Scripts
Software
Wastewater_10mL_Virus_Isolation
NAME
Software
Wastewater_10mL_Virus_Extraction
NAME
Before start
- Prepare 80% ethanol using 100% absolute ethanol and nuclease-free water, ensuring a minimum volume of 2 mL per sample.
- Vortex the Binding beads gently to ensure that the beads are fully resuspended.
- Prepare Binding Bead Mix — Combine 500 µL of Binding Solution with 20 µL of Binding Beads per sample, preparing enough for the required number of samples plus an additional 10% overage.
- Mix well by inversion, then store at room temperature.
Automated enrichment process
Automated enrichment process
Set up and label your plates according to the following table:
| Plate Name | Plate type | Contents | Vol per well | |
| Sample plate 1 | 24 deep-well | Clarified supernatant | 5 000 μL | |
| Dynabeads | 100 μL | |||
| Sample plate 2 | 24 deep-well | Clarified supernatant | 5 000 μL | |
| Lysis buffer | 24 deep-well | Lysis buffer | 500 μL | |
| Tip comb | 24 deep-well tip comb | Not applicable | ||
Select the appropriate program on the instrument (Wastewater_10mL_Virus_Isolation)
Start the run, then load the prepared sample and processing plates into position when prompted by the instrument
While the instrument is running, set up the nucleic acid extraction plates
Automated total nucleic acid extraction process
Automated total nucleic acid extraction process
Set up and label the Wash, Elution and Tip Comb plates outside of the instrument according to the following table:
| Plate name | Plate type | Component | Vol per well | |
| Wash Buffer 1 | 24 deep-well | Wash Buffer | 1 000 µL | |
| Wash Buffer 2 | 24 deep-well | 80% Ethanol | 1 000 µL | |
| Elution | 24 deep-well | Elution Solution | 100 µL | |
| Tip Comb | 24 deep-well tip comb |
When the enrichment process is complete (approximately 20 minutes after starting the run), remove the lysis plate from the instrument and store it safely for nucleic acid extraction. Discard the remaining plates from the instrument
Add 40 μL of Proteinase K to each enriched sample in the lysis plate
Invert the Binding Bead Mix tube several times to fully resuspend the beads, then add 520 μL to each sample in the lysis plate
Load the lysis plate along with Wash 1, Wash 2, Elution, and a new tip comb. Start the run on the instrument and select the appropriate programme (Wastewater_10mL_Virus_Extraction)
Immediately remove the Elution plate from the instrument at the end of the run and cover it. Alternatively, transfer the eluate to a new tube or plate for final storage. The isolated nucleic acid is ready for immediate use. For storage, keep it at –20°C for up to 6 months or at –80°C for longer than 6 months.
Manual enrichment process
Manual enrichment process
Follow the manufacturer's protocol available here: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0025695_MM_Wastewater_Ultra_NA_Isol_Kit_10mL_UG.pdf
Protocol references
This protocol is derived from Pub. No. MAN0025695 by Appliedbiosystems
Acknowledgements
This protocol is derived from Pub. No. MAN0025695 by Appliedbiosystems