Jul 02, 2026

ARISE Fungi DNA Extraction

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvoe1bxl4o/v1
  • 1Naturalis Biodiversity Center
Kevin Beentjes
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvoe1bxl4o/v1
Protocol Citation: Kevin Beentjes, Nafiesa Ibrahim, Tara van der Plas, Cas Lekkerkerker, Thirza van de Wetering, Emma Wubben 2026. ARISE Fungi DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoe1bxl4o/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: March 27, 2026
Last Modified: July 02, 2026
Protocol  Integer ID: 314016
Keywords: DNA extraction, fungi, magnetic bead, DNA purification
Funders Acknowledgements:
NWO Roadmap
Grant ID: 2020/ENW/00901156
Abstract
Magnetic bead DNA extraction with home-made chemicals, suitable for fungal (mushroom) samples.This protocol is designed as a low-cost, high-quality, high-throughput DNA extraction (ca € 0,20 per sample).
This protocol is the standard for fungal DNA extractions within the ARISE program at Naturalis Biodiversity Center.
For more information about ARISE, visit https://www.arise-biodiversity.nl/
Materials
Chemicals

  • Tris hydrochloride (1 M, pH 8)
  • EDTA (0.5 M, pH 8)
  • NaCl (5 M)
  • CTAB / cetyltrimethylammonium bromide

or instead of the above:
  • CTAB Lysis Buffer (Biochemica, A4150)

  • Milli-Q water
  • Tween 20
  • Guanidine hydrochloride
  • 100% absolute ethanol
  • 96.2% technical grade ethanol
  • Isopropanol
  • RNAse A (12mg/ml)
  • NaOH / 5M HCl to adjust pH
  • SeraSil-Mag 400 magnetic beads (Sigma-Aldrich, GE29357371)
Safety warnings
When preparing or handling chemicals, wear protective clothing: lab coats, gloves and safety glasses. Be aware of the dangers of each chemical, more information can be found in the relevant Safety Data Sheets (SDS).

Chemicals used in this protocol must be disposed of as hazardous chemical waste through an approved facility, complying with local and national regulations. Do not pour solutions down the drain, as it is harmful to aquatic life.

Do not add bleach or acidic solutions directly to the sample-preparation waste. Buffers containing guanidine thiocyanate or guanidine hydrochloride can form toxic gasses when combined with bleach.

CTAB is a harmful substance. Avoid skin contact. Please check the SDS for more information.
Preparation of Buffers
2% CTAB pH8 (per 100 ml)
  • Combine 10 ml 1 M Tris pH 8.0, 4 ml 0.5 M EDTA pH 8.0, 28.4 ml 5M NaCl and 2 g CTAB.
  • Add MilliQ water to a volume of 60 ml and heat in a water bath until CTAB is dissolved.
  • Bring the total volume to 100 ml with milliQ.

Ready-to-use CTAB buffer can also be purchased commercially.
15% Tween 20 (per 100 ml)
  • Add 15 ml of Tween 20 to 85 ml MilliQ.
  • Stir the mixture gently, don't create bubbles.
Tissue Binding Buffer (per 100 ml)
  • Dissolve 26.3 g guanidine hydrochloride in 20 ml milliQ.
  • Stir until dissolved.
  • Adjust the volume to 50 ml with milliQ.
  • Add 50 ml 100% ethanol.
Elution Buffer (per 100 ml)
  • Add 1 ml 1 M Tris HCl (pH 8) to 75 ml milliQ .
  • Add 0.2 ml 0.5 M EDTA (pH 8).
  • Bring to a final volume of 100 ml with milliQ.
  • Vortex/stir briefly to mix.
Calibration of Tissue Binding Buffer
  • Prepare a lysis mixture by mixing 4.54 mL CTAB, 330 µL Milli-Q and 330 µL 15% Tween.
  • Make a calibration solution by adding 10 ml Tissue Binding Buffer to the 5 ml prepared lysis mixture in a 50 ml tube.
  • Adjust to pH 4.0 - 4.5 with 5M HCl, while making sure the mixture is at room temperature and that it has been thoroughly mixed.
  • Take note of the volume 5M HCl added (this will be approximately 40 µl 5M HCl).
  • Dispose of the used calibration solution.
Take the desired volume of Tissue Binding Buffer and add the same volume 5M HCl added to the calibration solution for every 10 ml of tissue binding buffer (e.g. if 350 µl HCl was added to the 10 ml calibration solution, adjusting the pH of 100 ml of Tissue Binding Buffer would require the addition of 3.5 ml HCl).

Verify you got the right pH by adding 10 ml of binding buffer to 5 ml of fresh lysis mixture and measure the pH. Continue measuring and adjusting the pH as needed, the final combination of CTAB and Tissue Binding Buffer should be between pH 4.0 and 4.5. If a new buffers are made, the last verification step must be performed again.
Tissue Sampling and Lysis
Tissue Sampling
Note: For high throughput, DNA extraction can be performed in plates rather than individual tubes. Within ARISE fungal tissue samples are processed in Qiagen Collection Microtubes (Qiagen cat no. 19560) plates, which fit into a TissueLyser using plate adapters.

  • Prepare a plate of collection microtubes or 1.5 ml eppendorf tubes with 200 µl of CTAB buffer.
  • Place a small fragment of a mushroom (maximum around 5x5x5 mm) into a plate of collection microtubes or 1.5 ml eppendorf tubes.
  • For larger mushrooms, cut the fruiting body lengthwise and sample the flesh from the stem or cap. For smaller or slender fruiting bodies, take a fragment of the cap (including the hymenium). Very small specimens can be put into CTAB in their entirety.
Prepare Tissue Lysis Mix
  • Per sample, mix 18 μl of 15% Tween-20, 1 μl of Proteinase K, and 1 μl of RNase A.

Physical Lysis

  • Add 20 µl of prepared Tissue Lysis Mix per sample.
  • Add 5 to 10 sterile 1-2 mm glass beads per sample.

For optimal yield of DNA (but more fragmented, e.g. for DNA barcoding):
  • Use a TissueLyser for 3 minutes at 30 Hz.

For lower yield, but less fragmented DNA (e.g. for long-range PCR):
  • Use a TissueLyser for 20 seconds at 25 Hz.
  • Pause for 1 minute.
  • Repeat TissueLyser for 20 seconds at 25 Hz.
Chemical Lysis

  • Centrifuge for 2 minutes at max centrifuge speed to collect samples at the bottom of the plate / tube.
  • Incubate at 55°C, 400 RPM overnight on a shaking incubator.
  • Occasional vortexing can help to dissolve the tissue.
Possible stop. Store samples at 4 °C.
DNA Purification
Note: For high throughput, DNA extraction in plates can be automated using a purification instrument (e.g. a ThermoFisher Scientific KingFisher Flex or a MolGen PurePrep 96) or a liquid handling platform.

Binding

  • Centrifuge the incubated samples down for 5 minutes at max centrifuge speed at room temperature to pellet solid particles at the bottom of the plate / tube.
  • Transfer 75 µl of the supernatant to a deep well plate / tube.
  • Add 20 µl SeraSil-Mag 400 beads to each sample (make sure the beads are properly shaken into suspension before adding to the samples).
  • Add 150 μl (2x the lysate volume) of Tissue Binding Buffer to each sample.
  • Incubate 5 minutes at room temperature on a shaker.
Washing
  • Settle the beads on a magnetic rack and discard the supernatant.
  • Remove the plate / tube from the magnet and add 500 µl isopropanol.
  • Shake at room temperature at 500 rpm for 2 minutes.
  • Settle the beads on the magnet and discard the supernatant.
  • Remove the plate / tube from the magnet and add 400 µl 96% ethanol.
  • Settle the beads on the magnet and discard the supernatant.
  • Repeat last two steps to wash twice with 400 µl 96% ethanol.
  • Remove all ethanol and dry the beads for approximately 5-10 minutes at room temperature (or 50 °C).
Elution

  • Add 50-70 µl Elution Buffer (or MilliQ) to each sample and shake at 500 rpm for at least 5 minutes.
  • Settle the beads on the magnet and transfer the supernatant to a new plate / tube.
Protocol references
Partially based on the Mu-DNA protocol:
Sellers GS, Di Muri C, Gómez A, Hänfling B (2018) Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types. Metabarcoding and Metagenomics 2:24556.
https://doi.org/10.3897/mbmg.2.24556
We acknowledge support from the Dutch Research Council (NIEBA-ARISE project NWO Roadmap grant number 2020/ENW/00901156).