Apr 02, 2026
DNA extraction from fungal exsiccates/old fungarium collections
- Ursula Eberhardt1
- 1Eberhard-Karls-Universität Tübingen
Protocol Citation: Ursula Eberhardt 2026. DNA extraction from fungal exsiccates/old fungarium collections. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoe117l4o/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: April 01, 2026
Last Modified: April 02, 2026
Protocol Integer ID: 314340
Keywords: DNA barcoding, ITS, Sanger Sequencing, Genome skimming, herbarium, mushrooms, types, multilocus sequencing, fungi,
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Abstract
This protocol has been used extensively for the DNA extraction from fungarium material, mostly basidiomycete macrofungi, of different ages, using components of the Qiagen (Gentra) Puregene kits* for downstream PCR. The protocol includes adaptations to material of different ages and can be scaled easily. Because no columns or beads are used, large amounts of DNA can be retrieved. Typically, extracts have to be diluted for use in PCR. DNA extracts stored at -20°C for many years can still be used successfully for PCR.
Guidelines
- Use lab space and instruments dedicated to pre-PCR work.
- Aliquot all (kit) solutions.
- Use filter tips.
- If working with valuable material such as types or other old material, use fresh aliquots.
- Do not mix material of (too) different expected DNA contents. Older material is expected to have less DNA.
- Liquid transfers of solutions containing DNA are done by pouring rather pipetting.
- Inverting samples for 150 times or for 2 min may prevent DNA breakage compared to vortexing. However, a clear difference has not been observed in downstream PCR applications.
Materials
Qiagen Tissuelyzer with 24 tubes adapters
Lab centrifuge for 1.5 ml tubes
Heating/shaking block for 1.5 ml tubes
Pipettors and filter tips for 1-1000 ul
1.5 ml safe-lock centrifuge tubes; 2 tubes per sample
steel beads 2 mm, e.g. for ballbearings; 1 bead per sample
blotting paper/tissue paper
containers for aliquots (e.g. 50 ml single use plastic tubes or autoclaved glass bottles)
isopropanol
ethanol (high quality, non-degenerated) 70%
Qiagen Puregene kit components
DNA Lysis Solution
Protein Precipitation solution
DNA-hydration solution (10 mM Tris, 1 mM EDTA, pH 7-8)
Optional (old or scarce material)
glycogen solution (Qiagen)
Ultrapure water
For DNA extract diltutions for PCR applications
Ultrapure water
Optional:
Tube strips or 96-well plates
Multichannel pipettors
Troubleshooting
Safety warnings
- Special rules for DNA extraction and molecular work are likely apply when loaning material from public collections.
- Please be aware that Nagoya Protocol access restrictions to genetic resources may apply.
Sample Preparation
For each sample, prepare a 1.5 ml safe-lock centrifuge tube with a 2 mm steel bead.
Optionally, for each sample, another 1.5 ml safe-lock centrifuge tube with 300 μl isopropanol (step 5) can already be prepared, too.
For each sample, place a fragment of fungal tissue corresponding to a small crumb (from just visible dot to corresponding to 1-2 mm2 lamellae in a tube, using clean tools.
Sample disruption
Distribute tubes evenly between two 24 tube adapters of a Qiagen Tissuelyzer and disrupt material at maximum speed for three minutes. Extend disruption time if needed.
Lysis
Add 300 µl Cell Lysis Solution to each sample, opening only one tube at a time. Turn upside down for suspending powder sticking to the lid. Vortex to disperse powder in liquid.
(For old or scarce material)
Incubate overnight at 37 °C, optionally with shaking (minimum setting). Mix by inverting.
Incubate at 65 °C for 1 h, optionally with shaking (minimum setting).
For young, abundant material, incubation time can be reduced to 15 min.
Mix by inverting.
Protein precipitation
Cool samples to room temperature, e.g. by placing them in a freezer for a few minutes.
Add 100 μl Protein Precipitation Solution, mix by inverting 150 times or vortex briefly.
Note
- With certain types of racks it is possible to use two identical racks, one containing the tubes and another one as makeshift "lid" to invert up to 96 tubes at the same time.
Leave for at least 15 min at room temperature. Often, but not always, the liquid will turn milky.
DNA precipitation
For each sample, prepare a clean safe-lock tube with 300 μl isopropanol.
For old or scarce material, add into the lid of each tube 1 μl of glycogen solution, diluted 1:1 with ultrapure water.
Centrifuge samples for at least 5 min. Proceed immediately to next step.
Pour each sample in the prepared tube with isopropanol.
Note
- If the steel bead is transferred to the new tube by accident, remove it at once by sliding a stirring magnet along the outside of the tube towards the tube opening and discard the steel bead.
- The pelleted fungal fragments with or without the steel bead can be discarded or kept for a second DNA extraction, using the same or a different method.
Mix by inverting 150 times or vortexing.
(For old or scarce material)
Leave at room temperature or in the fridge overnight.
(For young material)
Leave at room temperature for 15 min.
Centrifuge samples for at least 5 min. Proceed immediately to next step.
DNA purification
Remove supernatant by pouring. Place each tube upside down on a prepared tissue for the remaining liquid to trickle down. The pellet is often, but not always, visible at the bottom of the tube.
Leave for a few minutes before blotting and turning around.
Add 300 μl 70% ethanol (high quality, not degenerated). Mix by inverting or vortexing.
Centrifuge samples for at least 5 min. Proceed immediately to next step.
Remove supernatant by pouring. Place each tube upside down on a prepared tissue for the remaining liquid to trickle down. Leave for not more than 10 min before proceeding to next step.
DNA hydration
Add 50 μl DNA hydration solution to each tube. Mix.
Place tubes for 1 h at 65 °C, optionally with gentle shaking.
Keep over night at room temperature before downstream use. Store at -20 °C or -80 °C for extended times.
Note
- Most DNA extracts are used diluted as PCR templates. A good starting point are dilutions of 1:25-1:50 for ribosomal genes and 1:25 for single copy genes, if using 10% template in a PCR reaction. Example: Add 1 μl DNA extract to 49 μl ultrapure water and use 2.5 μl diluted DNA extract in a 25 μl PCR reaction.
- If the PCR is unsuccessful, rather go for higher than lower dilutions.
- If dilutions are made in tube strips or 96-well plates, multichannel pipettors can be used for assembling the PCR reactions.
Protocol references
Qiagen (Jan 2022): PuregeneR DNA Handbook https://www.qiagen.com/en-US/resources/download/KitHandbook/puregene-dna-handbook, accessed 1 Apr 2026, and earlier editions.
QIAGEN Supplementary Protocol: Purification of archive-quality DNA from fungal tissue using the Gentra‱ Puregene‱ Tissue Kit or Gentra Puregene Mouse Tail Kit, https://www.qiagen.com/en-US/resources/download/Protocols/pg23-purification-of-archive-quality-dna-from-fungal-tissue-using-the-gentra-puregene-tissue-kit-or, accessed 1 Apr 2026
The protocol as described here has, for example, been used in for older fungal collections:
U. Eberhardt, N. Schütz, P. Bartlett, and H. J. Beker, “96 North American taxa sorted – Peck’s Hebeloma revisited,” Mycologia, vol. 114, no. 2, pp. 337–387, Mar. 2022, doi: https://doi.org/10.1080/00275514.2021.2012063
Another version of this protocol has been used in:
C. Cripps, U. Eberhardt, N. Schütz, H. J. Beker, V. S. Evenson, and E. Horak, “The genus Hebeloma in the Rocky Mountain alpine zone,” MycoKeys, vol. 46, pp. 1–54, 2019, doi: https://doi.org/10.3897/mycokeys.46.32823.
Acknowledgements
N. Schütz is acknowledged for devising the protocol version used in Cripps et al. (2019).