Aug 14, 2025
Whole-Genome DNA Extractions for MinION Sequencing Using the Macherey-Nagel NucleoBond HMW DNA Kit
- Stephanie Le Prieur1,
- Anne Genissel2
- 1CNRS, Universite Paris-Saclay;
- 2INRAE, Universite Paris-Saclay
- Evolmol-protocols
Protocol Citation: Stephanie Le Prieur, Anne Genissel 2025. Whole-Genome DNA Extractions for MinION Sequencing Using the Macherey-Nagel NucleoBond HMW DNA Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg31x5pl25/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 14, 2025
Last Modified: August 14, 2025
Protocol Integer ID: 224692
Keywords: fungal genome, genome assembly of natural isolate, genome dna extractions for minion, fungal ascomycete zymoseptoria tritici, genome dna extraction, genomic dna extraction protocol, nagel nucleobond hmw dna kit long, genome assembly, weight dna suitable for both pacbio, ascomycete zymoseptoria tritici, fungal, read sequencing, genome, oxford nanopore technology, weight dna, sequencing platform
Abstract
Long-read sequencing is increasingly accessible, enabling unprecedented insights into the pervasive structural rearrangements of fungal genomes. In this study, we optimized a genomic DNA extraction protocol to obtain unsheared, high-molecular-weight DNA suitable for both PacBio and Oxford Nanopore Technologies sequencing. We validated the protocol with both sequencing platforms, achieving in both case telomere-to-telomere genome assembly of natural isolates of the fungal ascomycete Zymoseptoria tritici.
Guidelines
18. Precipitate the DNA by adding 3.5 mL isopropanol to each eluate. Mix gently by inversion 10 times (a DNA pellet should appear).
19. Centrifuge for 10 min at 4,500 g.
20. Carefully discard the supernatant by pipetting without losing the DNA pellet.
21. Add 2 mL of 70% ethanol (0,22uM filter-sterilized) to each DNA pellet.
22. Centrifuge for 5 min at 4,500 g.
23. Carefully discard the supernatant.
24. Dry the pellet at room temperature (ensure no ethanol remains, but do not over-dry as it will make resuspension difficult).
25. Resuspend the DNA in 150 µL HE buffer by flicking the tube or gently pipetting the pellet (use wide-bore tips!).
26. Transfer the DNA to a 1.5 mL microtube. If the liquid is still cloudy, add 50–100 µL (or more) HE buffer.
Quality control using Nanodrop, Qubit and agarose gels.
Materials
Fungal sample preparation:
- Petri dishes with PDA medium
- Eppendorf tubes (2 mL)
- Parafilm (for sealing)
- Retsch grinder and metal beads
- Macherey-Nagel NucleoBond HMW DNA Kit (including buffers H1, H2, H3, H4, H5, RNase A, proteinase K)
- 15 mL polypropylene conical centrifuge tubes
- 2 mL microtubes
- 50 mL tubes
- Isopropanol (for DNA precipitation)
- 70% ethanol (0.22 µM filter-sterilized)
- HE buffer
- Wide-bore pipette tips
- Nanodrop, Qubit, and agarose gels (for quality control)
Troubleshooting
Fungal sample preparation
Grow strains on Petri dishes with PDA medium for 4 days at 20 °C.
Scrape the plates and place them into 2 mL Eppendorf tubes, store at –80 °C for at least 24 h, then lyophilize for 2 days (opened tube and sealed with pierced parafilm).
Grind lyophilized mycelium using the Retsch grinder and metal beads for 40 sec × 2 at 30 Hz. The powder should be fine.
Weigh out 250 mg of dried mycelium for DNA extraction.
Macherey-Nagel HMW protocol steps
Add 900 µL of lysis buffer H1 + 200 µL proteinase K to each sample.
Vortex for 2 min.
Centrifuge for 2 min at 11,000 G.
Transfer sample to a 15 mL tube and add 4 mL of buffer H1.
Aliquot the sample into three 2 mL microtubes, then vortex for 5 sec.
Incubate samples at 50 °C for 30 min.
Combine the aliquots back into one new 15 mL polypropylene, conical centrifuge tube and add 100 µL RNase A. Mix gently by inverting the tube and incubate for 5 min at room temperature.
Place the NucleoBond HMW column on a 50 mL tube using the plastic rings from the manufacturer. Equilibrate with 12 mL buffer H2 by pouring along the sides of the filter (not at the center!). Let it drain by gravity.
Discard the flow-through.
Add 10 mL buffer H2 to each sample tube and mix gently by inversion.
Load the sample onto the center of the column and let it flow through by gravity.
Discard the flow-through.
Add 6 mL buffer H3 to the column (along the sides!), let it flow through.
Discard the flow-through and the column filter.
Wash the column (without filter) with 12 mL buffer H4.
Discard the flow-through.
Place the column on new 50 mL tubes for DNA elution. Add 5 mL of elution buffer H5.
Precipitate the DNA by adding 3.5 mL isopropanol to each eluate. Mix gently by inversion 10 times (a DNA pellet should appear).
Centrifuge for 10 min at 4,500 g.
Carefully discard the supernatant by pipetting without losing the DNA pellet.
Add 2 mL of 70% ethanol (0,22uM filter-sterilized) to each DNA pellet.
Centrifuge for 5 min at 4,500 g.
Carefully discard the supernatant.
Dry the pellet at room temperature (ensure no ethanol remains, but do not over-dry as it will make resuspension difficult).
Resuspend the DNA in 150 µL HE buffer by flicking the tube or gently pipetting the pellet (use wide-bore tips!).
Transfer the DNA to a 1.5 mL microtube. If the liquid is still cloudy, add 50–100 µL (or more) HE buffer.
Quality control using Nanodrop, Qubit and agarose gels.