Feb 04, 2026
Protocol for environmental Nucleic Acid (RNA and DNA) extraction from filter sample using MagMAX Microbiome Ultra Nucleic Acid Isolation Kit
- Yoko Makabe-Kobayashi1,
- Koji Hamasaki1
- 1Atomosphere and Ocean Research Institute, The University of Tokyo
Protocol Citation: Yoko Makabe-Kobayashi, Koji Hamasaki 2026. Protocol for environmental Nucleic Acid (RNA and DNA) extraction from filter sample using MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl88jp7l2w/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 06, 2025
Last Modified: February 04, 2026
Protocol Integer ID: 224228
Keywords: DNA extraction, MagMAX, microbe, water, filter, Sterivex, environmental Nucleic Acid, RNA Extraction, Microbiome Ultra Nucleic Acid Isolation Kit, eDNA, eRNA, magmax microbiome ultra nucleic acid isolation kit, microbiome ultra nucleic acid isolation kit the objective, microbiome ultra nucleic acid isolation kit, protocol for environmental nucleic acid, protocol for ena extraction, environmental nucleic acid, environmental rna, microbial community analysis, ena extraction, microbe ena, extraction from water sample, molecular biology analysis, extraction from filter sample, rna, µm sterivextm filter unit, water sample, extraction, filter sample
Abstract
The objective of this protocol is environmental RNA and DNA (eNA) extraction from water samples filtered through 0.22 µm SterivexTM filter units or polycarbonate 3.0 µm filter, using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit (Thermo Fisher Scientific)
It was modified by based on a previously established and standardized our protocol for eNA extraction for microbial community analysis.
This protocol is used upstream to molecular biology analysis (e.g. NGS, cDNA library construction, RT-PCR, qPCR) to specifically target microbe eNA.
Guidelines
This protocol has been modified Manual provided by Thermo Fisher Scientific:
- MagMAX‱ Microbiome Ultra Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Catalog Number A42358)(with tube, 100 preps));
MagMAXMicrobiomeNuclAcidIsolatKit_SoilSalivaUrine_Manually_UG.pdf1.1MB Materials
- Samples
- Sterivex-GP 0.22 μm cartridges (Merck Millipore, SVGP01050) :
For both RNA and DNA samples, cartridges were filled with 1.6 mL of RNAlater‱ Stabilization Solution (Invitrogen‱, AM7020) and stored at −20 °C.
For DNA-only samples, cartridges were stored without RNAlater at −80 °C.
- Nuclpore membranes (PC MB, 47 mm, 3.0 μm) :
For both RNA and DNA samples, membranes were treated with RNAlater‱ Stabilization Solution after that stored at −20 °C.
For DNA-only samples, membranes were stored without RNAlater at −80 °C.
- Reagents
- MagMAX‱ Microbiome Ultra Nucleic Acid Isolation Kit [with tubes] (Thermo Fisher Scientific, A42358)
- RNase AWAY (Thermo Fisher Scientific, 7002)
- Nuclease-Free Water: Distilled Water, Deionized, Sterile (NIPPON GENE, 316-90101)
- Ethanol(99.5) for Molecular Biology (FUJIFILM, 054-07225)
- Equipments
- Designated RNA work area and Biological Safety Cabinet
- Microsmash Beads Beater (TOMY, MS-100)
- Microcentrifuge for 5 mL and 2mL tubes : Centrifugal force needed: 5000 x g
- Digital VORTEX-GENIE 2 (Scientific Industries, SI-A236)
with 6-inch Platform (Scientific Industries, 146-6005-00) and Microtube Foam Insert (Scientific Industries, 504-0234-00)
- DynaMag-2 (Thermo Fisher Scientific, DB12321)
- Aluminum block thermostatic bath for 2mL tubes : 75℃
- Sterile forceps : For transfering the filter into a 5 mL tube
- Pipettes : 1000μL - 100μL
- Consumables
- Filter tips : 1000μL - 100μL
- 5 mL tubes (TOMY, TM-657S)
- 2 mL tubes (VIOLAMO, 1-1600-04)
- 1.5 mL tubes
- Terumo Syringes 10mL for Vaccination Slip Tip White (TERUMO, SS-10SZ) : For pushing
out the RNAlater from the Sterivex cartridge
Troubleshooting
Before start
- Wear gloves throughout the extraction process.
- Use filter tips to minimize the risk of contaminations.
- All steps have to be conducted within a designated RNA work area.
a) Turn on Biological Safety Cabinet blower. Clean the work surfaces with RNase AWAY (Thermo Fisher Scientific, 7002) to remove nucleases. Then Lower the sash and run the UV light for 30 minutes. Switch the UV light back to white light; you are ready to begin.
b) Precipitates can occur if the Lysis Buffer or Binding Solution is stored when room temperature is too cold. If there are precipitates, warm the reagents at 37°C and gently mix to dissolve the precipitates. Avoid creating bubbles.
c) Prepare 80% Ethanol from 99.5% absolute Ethanol and Nuclease-Free Water. Prepare enough solution for a minimum volume of 2 mL per sample.
Before each use of the kit
1. Vortex the Beads vigorously to ensure they are homogenous.
2. Prepare Binding/Bead Mix according to the following table:
| Component | Volume per well [1] |
|--------------------------------------|---------------------|
| Total Nucleic Acid Binding Buffer | 500 µL |
| Total Nucleic Acid Magnetic Beads | 20 µL |
|--------------------------------------|---------------------|
| Total volume | 520 µL |
[1] Use 10% overage calculation when making a master mix for use with multiple samples.
3. Mix well by inversion, then store at room temperature.
Transfer the Sample into the beads tube
Push out the RNAlater from the Sterivex cartridge with 10 mL syringe (TERUMO, SS-10SZ).
Open the Sterivex cartridg by a tube cutter. Using a surgical scalpel (Akiyama MEDICAL MFG, Replacement blade No. 15) and two sets of sterile forceps, pick up the filter membrane at opposite edges and transfer the filter into a 5 mL tube (TOMY, TM-657S) with the top side inward.
Add the homogenization beads (provided in the kit) into the 5 mL tube.
Sample lysis
7m 30s
Add 800 µL of the Lysis Buffer to the 5 mL tubes.
800 µL
Place the 5 mL tubes into Microsmash Beads Beater (TOMY, MS-100). Shake at speed 3,800 rpm for 1 minute and then rest for 30 seconds, then 3,800 rpm for 1 minute.
3800 rpm, Room temperature , 00:01:00 00:00:30 3800 rpm, Room temperature , 00:01:00
Equipment
Microsmash Beads Beater
NAME
TOMY
BRAND
MS-100
SKU
LINK
2m 30s
Centrifuge the 5 mL tubes at 5000×g for 5 minutes at room temperature.
5000 rpm, Room temperature, 00:05:00
5m
Transfer the supernatant to a clean 2 mL Tube (VIOLAMO MicroTube 1-1600-04). It will be necessary to push the tip of pipett through the beads into the bottom of the tube in order to recover as much supernatant as possible.
Note: However, don't transfer the beads as much as possible.
<STOPPING POINT> Bead tube can be stored at 4°C overnight after lysis.
Bind the Magnetic Beads
10m
Invert the Binding Bead Mix to mix, then add 520 µL to each tubes.
520 µL
Note: Remix the Binding Bead Mix by inversion frequently during pipetting to ensure even distribution of beads to all samples or wells. The mixture containing the Binding Beads is viscous. Therefore, pipet slowly to ensure that the correct amount is added. DO NOT use a repeat pipet to add to the samples as the high viscosity will cause variations in volume added.
Note: Centrifuge the tube at 800 rpm for 1 minute to collect liquid to bottom of tube if needed.
Shake the tube at 900 rpm for 5 minutes.
900 rpm, Room temperature , 00:05:00
Equipment
Digital VORTEX-GENIE 2
NAME
Scientific Industries, Inc
BRAND
SI-A236
SKU
LINK
Equipment
Tube Support for Vortex GENIE2
NAME
Scientific Industries, Inc.
BRAND
504-0234-00
SKU
Equipment
6 Platform Head, for Vortex GENIE2
NAME
Scientific Industries, Inc.
BRAND
146-6005-00
SKU
5m
Place the tube on the magnetic stand for at least 5 minutes, or until all the beads have collected00:05:00 Room temperature
Equipment
DynaMag™-2 Magnet
NAME
Magnetic tube rack
TYPE
Invitrogen™
BRAND
12321D
SKU
LINK
5m
Wash the Magnetic Beads
24m
Keeping the tube on the magnet, carefully open the lid, then discard the supernatant.
Note: Be sure to completely remove any contaminating homogenization beads.
IMPORTANT! Avoid disturbing the magnet beads.
Remove the tube from the magnetic stand, then add 1 mL of the Wash Buffer to each sample.
1 mL
Shake at 800 rpm for 30 seconds.
800 rpm, Room temperature , 00:00:30
Equipment
Digital VORTEX-GENIE 2
NAME
Scientific Industries, Inc
BRAND
SI-A236
SKU
LINK
Equipment
Tube Support for Vortex GENIE2
NAME
Scientific Industries, Inc.
BRAND
504-0234-00
SKU
Equipment
6 Platform Head, for Vortex GENIE2
NAME
Scientific Industries, Inc.
BRAND
146-6005-00
SKU
30s
Place the tube on the magnetic stand for 3 minutes, or until all the beads have collected.
00:03:00
Equipment
DynaMag™-2 Magnet
NAME
Magnetic tube rack
TYPE
Invitrogen™
BRAND
12321D
SKU
LINK
3m
Keeping the tube on the magnet, carefully open the lid, then discard the supernatant from each well.
IMPORTANT! Avoid disturbing the magnet beads.
Repeat from step 12 to step 15 using 1 mL of the Wash Buffer.
1 mL 00:03:30
3m 30s
Repeat from step 12 to step 15 using 1 mL of 80% Ethanol.
1 mL 00:03:30
3m 30s
Repeat from step 12 to step 15 using 1 mL of 80% Ethanol.
1 mL 00:03:30
3m 30s
Dry the beads with open the cap for 10 minutes at room temperature.
00:10:00 Room temperature
10m
Elute the nucleic acid
13m
Add 50 µL of the Elution Solution to each tubes.
50 µL
Place the tube on heat-block at 75°C for 5 minutes.
75 °C 00:05:00
5m
Place the tube on a shaker at 800 rpm for 5 minutes.
800 rpm, Room temperature , 00:05:00
Equipment
Digital VORTEX-GENIE 2
NAME
Scientific Industries, Inc
BRAND
SI-A236
SKU
LINK
Equipment
Tube Support for Vortex GENIE2
NAME
Scientific Industries, Inc.
BRAND
504-0234-00
SKU
Equipment
6 Platform Head, for Vortex GENIE2
NAME
Scientific Industries, Inc.
BRAND
146-6005-00
SKU
5m
Place the tube on the magnetic stand for 3 minutes or until all beads are collected against the magnets.
00:03:00
Equipment
DynaMag™-2 Magnet
NAME
Magnetic tube rack
TYPE
Invitrogen™
BRAND
12321D
SKU
LINK
3m
Keep the tube on the magnet and carefully open the lid, then transfer the eluates to a new 1.5 mL tube.
The purified nucleic acid is ready for immediate use. Alternatively, store at −20°C for up to 6 months and −80°C for greater than 6 months.
Options
For DNA sample: Phenol-chloroform extraction (PCI purification) is performed after RNase treatment.
For RNA sample: TURBO DNA-free‱ Kit (Invitrogen, AM1907) is performed.