Mar 03, 2026

Public workspaceHigh-Quality Extraction of Medium-to-Long DNA Fragments from Biogas Digestate for Nanopore Sequencing

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld67bng5b/v1
  • He Sun1,
  • Anna Schnürer1
  • 1Department of Molecular Sciences, Swedish University of Agricultural Sciences.
H S.
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld67bng5b/v1
Protocol Citation: He Sun, Anna Schnürer 2026. High-Quality Extraction of Medium-to-Long DNA Fragments from Biogas Digestate for Nanopore Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld67bng5b/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: September 17, 2025
Last Modified: March 03, 2026
Protocol Integer ID: 227535
Keywords: biogas digestate, DNA extraction, High-quality DNA, HMW DNA, long dna fragments from biogas digestate, biogas digestate sample, biogas digestate, optimized dna extraction protocol, dna extraction protocol, different biogas plant, long dna fragment, nanopore, dna of sufficient purity, dna, read sequencing
Funders Acknowledgements:
Ekhagastiftelsen
Grant ID: 2024-117
Abstract
This optimized DNA extraction protocol is designed to obtain high-quality, medium-to-long DNA fragments from biogas digestate for Nanopore sequencing. The procedure employs the Qiagen DNeasy PowerSoil Pro Kit and consistently yields DNA of sufficient purity (A260/280 = 1.7–1.9; A260/230 > 2.0), fragment length (5–20 kb), and concentration suitable for long-read sequencing. The protocol has been validated on ten biogas digestate samples derived from different biogas plants, demonstrating robust and reproducible performance.
Guidelines
  • Ensure that the PowerBead Pro Tubes rotate freely in the centrifuge without rubbing. 
  • If Solution CD3 has precipitated, heat at 60°C until precipitate dissolves. 
  • Perform all centrifugation steps at room temperature (15–25°C). 
Materials



NameCATALOGVENDOR
HighPrep PCRMAGBIO
DNeasy PowerSoil Pro Kit4701450QIAGEN
Ethanol
Nuclease-free water

Troubleshooting
Before start
Turn on the heating plate and set it to 37 °C.
Sample Preparation
5m
Decant the beads from the PowerBead Pro Tube into a new 1.5 mL Microcentrifuge Tube.

Weigh the empty PowerBead Pro Tube and record the weight.
Using a cut 1000 μl pipette tip, transfer 600 μl + 600 μl of homogenized digestate sample into the empty PowerBead Pro Tube.
Amount1200 µL Digestate Sample

Centrifuge the tube at 10,000 × g for 5 minutes.
Centrifigation10000 x g, 20°C, 00:05:00

5m
Carefully decant the supernatant, weigh the PowerBead Pro Tube again, and record the weight.
Note: Calculate the sample weight.
Decant the beads back into the PowerBead Pro Tube.
DNA Extraction
8m 50s
Add 800 μl of Solution CD1. Vortex briefly to mix.
Amount800 µL Solution CD1

Homogenize via FastPrep‱-24 Classic I at 6 m/s for 20 s. Immediately place the tube on ice after lysis.
Duration00:00:20
The homogenization parameters may be adjusted according to sample type.
20s
Centrifuge the PowerBead Pro Tube at 15,000 x g for 2 min.
 Centrifigation15000 x g, 20°C, 00:02:00

2m
Carefully decant the supernatant into a new 2.0 mL Microcentrifuge Tube.
Repeat step 9.
2m
Transfer the supernatant to a clean 2 mL Microcentrifuge Tube.
Note: Expect 500- 600 μl. The supernatant may still contain some soil particles. 
Add 200 μl of Solution CD2 to the tube and mix thoroughly by gently inverting for 1 min.
Amount200 µL CD2 Duration00:01:00
1m
Centrifuge at 15,000 x g for 90 s. Carefully transfer up to 700 µL of the supernatant into a clean 2.0 mL Microcentrifuge Tube, avoiding disturbance of the pellet.
Note: Expect 500- 600 μl.
Centrifigation15000 x g, 20°C, 00:01:30

1m 30s
Repeat step 12, centrifuging for 60 s instead.
Note: Almost all of the liquid can be transferred. A pellet mark may be visible on the tube wall.
Centrifigation15000 x g, 20°C, 00:01:00

1m
Add 600 μl of Solution CD3 and mix thoroughly by gently inverting for 1 min.
Amount600 µL CD3 Duration00:01:00

Note
If DNA purification is planned, bring the magnetic beads to equilibrate to room temperature at this step.

1m
DNA Cleaning
6m
Load 650 μl of lysate to an MB Spin Column. Centrifuge at 8,000 x g for 1 min. 
Centrifigation8000 x g, 20°C, 00:01:00

1m
Discard the flow-through and repeat step 14 to ensure that all of the lysate has passed through the MB Spin Column. 
Centrifigation8000 x g, 20°C, 00:01:00
1m
Carefully place the MB Spin Column into a clean 2 mL Collection Tube. Avoid splashing any flow-through onto the MB Spin Column. 
Add 500 μl of Solution EA to the MB Spin Column. Centrifuge at 8,000 x g for 1 min. 
Amount500 µL Solution EA
Centrifigation8000 x g, 20°C, 00:01:00

1m
Discard the flow-through and place the MB Spin Column back into the same 2 mL Collection Tube. 
Add 500 μl of Solution C5 to the MB Spin Column. Centrifuge at 8,000 x g for 1 min. 
Amount500 µL Solution C5
Centrifigation8000 x g, 20°C, 00:01:00
1m
Discard the flow-through and place the MB Spin Column into a NEW 2 ml Collection Tube. 
Centrifuge at up to 15,000 x g for 2 min. Carefully place the MB Spin Column into a new 2.0 mL Microcentrifuge Tube.
Centrifigation15000 x g, 20°C, 00:02:00
2m
DNA Elution
8m
Add 62 μl of Solution C6 to the center of the white filter membrane. Incubate for 5 min.
Amount62 µL Solution C6
Duration00:05:00

5m
Centrifuge at 8,000 x g for 3 min. Discard the MB Spin Column. The DNA is now ready for downstream applications.
Centrifigation8000 x g, 20°C, 00:03:00
Note
Use 1.5 μl of the eluate to assess the DNA quality and estimate concentration via Nanodrop.


3m
DNA Purification
20m 30s
Add 21 µL of magnetic beads (0.35× volume; e.g., AMPure or HighPrep) to 60 µL of DNA sample. Mix thoroughly by gentle hand shaking and flicking until the beads are evenly suspended.
Amount60 µL DNA Sample
Amount21 µL Magnetic Beads
Note: Bring the beads to room temperature for at least 30 min before use.

Incubate the mixture at room temperature for 10 minutes.
Duration00:10:00

10m
Briefly centrifuge the tube, then place it on a magnetic stand until the solution becomes clear and the beads are fully bound.
While the tube remains on the magnet, carefully remove and discard the supernatant without disturbing the bead pellet.
(wash 1/2) Add 200 µL of freshly prepared 80% ethanol. Avoid disturbing the beads. Incubate for 30s.
Amount200 µL Fresh 80% Ethanol
Duration00:00:30

30s
(wash 1/2) Remove the supernatant.
Repeat steps 28-29.

Note
If the final DNA purity is insufficient, perform up to three wash steps. Note that excessive washing may lead to increased DNA loss.

Briefly centrifuge the tube, place it on the magnetic stand, and remove any residual liquid with a 10 µL pipette.
Air-dry the beads for 1–2 minutes, avoiding over-drying or cracking of the pellet.
Add 30 µL of nuclease-free water and resuspend the beads gently by shaking and flicking until fully dispersed.
Amount30 µL Nuclease-free water

Incubate at 37 °C for 10 minutes.
Duration00:10:00
Temperature37 °C

10m
Briefly centrifuge the tube, then place it on the magnetic stand until the solution is clear and the beads are fully bound.
Collect the supernatant, which contains the purified DNA.
Amount30 µL DNA Eluate
Note
  • Use 1.5 µL of the eluate to assess DNA purity with a NanoDrop spectrophotometer.
  • Use 2 µL to determine DNA concentration with a Qubit fluorometer.
  • Use 5 µL to evaluate DNA fragment length by agarose gel electrophoresis.


For short-term storage, keep the extracted DNA at 4 °C and proceed with sequencing library preparation as soon as possible.