Apr 24, 2026
Version 2
Optimized QIAGEN DNeasy Plant Pro Kit Protocol for Fungi gDNA Extraction V.2
- Cyrine ABID1,2,
- Hela Zouari-Mechichi3,4,
- Tahar Mechichi3,
- Najla Kharrat5
- 1Laboratory of Molecular and Cellular Screening Process, Centre of Biotechnology of Sfax, University of Sfax, PO box 1177, Sfax 3018, Tunisia;
- 2National School of Engineers of Sfax, University of Sfax, Sfax 3038, Tunisia;
- 3Laboratory of Biochemistry and Enzymatic Engineering of Lipases, Ecole Nationale d’Ingénieurs de Sfax (ENIS), University of Sfax;
- 4Institut Supérieur de Biotechnologie de Sfax, University of Sfax, Tunisia;
- 5Laboratory of Screening Cellular and Molecular Process, Centre of Biotechnology of Sfax, University of Sfax, PO box 1177, Sfax 3018, Tunisia
- Cyrine ABID: PhD Student;
Protocol Citation: Cyrine ABID, Hela Zouari-Mechichi, Tahar Mechichi, Najla Kharrat 2026. Optimized QIAGEN DNeasy Plant Pro Kit Protocol for Fungi gDNA Extraction . protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r6yjg1b/v2Version created by Cyrine C.A ABID
Manuscript citation:
MDPI and ACS Style
Abid, C.; Zouari-Mechichi, H.; Benmarzoug, R.; Mechichi, T.; Kharrat, N. Impact of DNA Extraction Strategies on Genomic and Bioinformatic Outcomes in Eight Selected Fungal Strains. J. Fungi 2026, 12, 299. https://doi.org/10.3390/jof12050299
AMA Style
Abid C, Zouari-Mechichi H, Benmarzoug R, Mechichi T, Kharrat N. Impact of DNA Extraction Strategies on Genomic and Bioinformatic Outcomes in Eight Selected Fungal Strains. Journal of Fungi. 2026; 12(5):299. https://doi.org/10.3390/jof12050299
Chicago/Turabian Style
Abid, Cyrine, Hela Zouari-Mechichi, Riadh Benmarzoug, Tahar Mechichi, and Najla Kharrat. 2026. "Impact of DNA Extraction Strategies on Genomic and Bioinformatic Outcomes in Eight Selected Fungal Strains" Journal of Fungi 12, no. 5: 299. https://doi.org/10.3390/jof12050299
APA Style
Abid, C., Zouari-Mechichi, H., Benmarzoug, R., Mechichi, T., & Kharrat, N. (2026). Impact of DNA Extraction Strategies on Genomic and Bioinformatic Outcomes in Eight Selected Fungal Strains. Journal of Fungi, 12(5), 299. https://doi.org/10.3390/jof12050299
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 23, 2026
Last Modified: April 24, 2026
Protocol Integer ID: 315613
Keywords: gDNA, Optimization, Extraction, Fungi, Mycelium, Mushroom, Quality, fungi gdna extraction, gdna extraction from diverse fungal sample, gdna extraction from plant, gdna extraction, gdna quality, optimized qiagen dneasy plant pro kit protocol, diverse fungal sample, gdna, ascomycota fungi phyla, mycelium liquid culture, downstream genomic analysis, supporting downstream genomic analysis, basidiomycota, genomic dna, manual extraction, extracted sample, qiagen, dneasy plant pro kit, mushroom tissue, repeatability in the manual extraction
Funders Acknowledgements:
NGS-4-ECOPROD Project "From Next Generation Sequencing Microorganisms Towards Ecofriendly Biotech Based Products" which has received funding from the European’s Union’s Horizon Europe Research and Innovation Program
Grant ID: Grant Agreement ID n°101079425
Abstract
This protocol describe the optimisation of a commercial kit "DNeasy Plant Pro Kit, QIAGEN" reserved to gDNA extraction from plant.
This protocol provides clear, straightforward, and transparent mandatory steps to ensure reproducibility and repeatability in the manual extraction of genomic DNA (gDNA) from mycelium liquid cultures or mushroom tissues. It has been successfully tested across various species belonging to the Basidiomycota and Ascomycota fungi phyla.
To evaluate gDNA quality, multiple validation steps were applied to the extracted samples.
To evaluate gDNA quality, multiple validation steps were applied to the extracted samples. The protocol was effectively employed by Cyrine et al. 2026 (https://doi.org/10.3390/jof12050299) for gDNA extraction from diverse fungal samples, supporting downstream genomic analyses.
Guidelines
Adhere to standard molecular biology techniques, including:
- Always use filter tips to minimize contamination risks.
- Change gloves frequently, especially when contamination is suspected.
- Keep freshly prepared 10% bleach and 70% ethanol ready-to-use solutions available for decontaminating surfaces and equipment.
Materials
‱ Reagents
DNeasy Plant Pro Kit (50)QiagenCatalog # 69204
Ethanol absolute ≥99.8%VWR International (Avantor)Catalog #20821.365
Ultrapure water and autoclaved for the wash
Water, Ultra Pure, Free of DNase, RNase, Protease, EndonucleaseBio Basic Inc.Catalog #41116113
Lliquid nitrogen and container
‱ Equipment
• Porcelain mortar with pestle;
• Sterile scalpel or sterile scissors;
‱ Laboratory Equipment and Consumables
• -80°C Freezer (For long-term storage of gDNA and samples);
• -20°C Freezer (For short and medium term storage of gDNA and samples);
• Laboratory Ice Buckets/Trays;
• Ice;
• FinntiP Flex 1000 (ThermoFisherScientific, cat.no.94060 )
• FinntiP Flex 200 (ThermoFisherScientific, cat.no.94060310)
• FinntiP Flex 10 (ThermoFisherScientific, cat.no.94060100)
• Petri-dishes 60mm;
• Individual 12-ply Sterile Medical gauze pads (10x10cm; 100% Cotton);
• Freeze-dryer for mushroom samples;
Other materials such as parafilm, nitrile gloves, racks, pipette filter tips, centrifuge, vortex etc. are general use and the exact brand selected should not impact results.
Protocol materials
DNeasy Plant Pro Kit (50)QiagenCatalog # 69204
Ethanol absolute ≥99.8%VWR International (Avantor)Catalog #20821.365
Ultrapure water
Water, Ultra Pure, Free of DNase, RNase, Protease, EndonucleaseBio Basic Inc.Catalog #41116113
Lliquid nitrogen and container
UltraPure™ DNase/RNase-Free Distilled WaterThermo FisherCatalog #10977015
Safety warnings
Buffer APP contains guanidine thiocyanate
Buffer AW1 contains guanidine hydrochloride
Before start
From the QIAGEN DNeasy Plant Pro Kit Handbook (version 2019, page12):
Important points before starting
-1- Ensure that the tissue disruption tubes rotate freely in the centrifuge, without rubbing against
the sides.
-2- Perform all centrifugation steps at room temperature (15–25°C).
-3- Buffer AW1 and Buffer AW2 are supplied as concentrates. Add ethanol (96–100%) according to the bottle label before use to obtain a working solution.
-4- If Buffer APP has precipitated, heat at 60°C until precipitate dissolves
Preperation of Fungal Sample
1w 1d 0h 22m
- Case n°1: Extraction from Mycelia Culture
3d 0h 17m
Collect sterilely the grown mycelia from the culture media in a Petri-dish;
50 mL Mycelia Culture
2m
Place it immediately at -20°C for 12-24h;
24:00:00 Time -20 °C Freezer
Note
Note 1: In case of need to transport from laboratory to another, the material should be transported in a cold and isothermal environment.
Note 2: it is possible to store the sample at -20°C short term or -80°C long term until use.
1d
Using a sterile medical pads, the thawed sample is rinsed with 3ml autoclaved ultrapure water every sample was rinsed with autoclaved ultrapure water (≈3ml), and then manually squeezed
Note
Note 1: The sample should be thawed at room temperature for maximum 90min before use
Note 2: The biomass is rinsed with water by adding 1 ml of each separately, using P1000, while spreading it over the entire surface of the biomass.
Note 3: Operate quickly
10m
Place the grown, rinsed, and squeezed mycelia at -20°C for 48h;
48:00:00 Time -20 °C Freezer
Note
Note 1: Store the porcelain morter and pestle at 20°C for at least 24 hours to prepare to the next step
Note 2: it is possible to store the sample at -20°C short term or -80°C long term until use.
2d
Transfer the grown, rinsed, squeezed, and froozen mycelia into fine power due to liquid nitrogen treatment using a porcelain mortar with pestle.
Note
The mechanical action should be performed in a cold environment as mentioned in the figure below
5m
Place the obtained fine powder at -20°C or -80°C until use.
- Case n°2: Extraction from Fresh Collected Mushroom
5d 0h 5m
Extract the intern tissue of the fresh fruiting mushroom body (Stalk or Cap) with a sterile scalpel. If it is not possible (tissue criteria), cut the mushroom tissue into small pieces using sterile scissors;
5m
Place it at -20°C for 48h;
48:00:00 Time -20 °C Freezer
2d
Freeze-dring the frozen mushroom sample for 1 cycle of 24h. Stock it in -20°C or -80°C until use.
1d
Place 100-300 mg of fungi (mycelia or mushroom) fine powder, treated with liquid
nitrogen or lyophilization treament respectively, to a 2 ml tissue disruption tube at -20°C for 48h;
100-300 mg Fine Powder of the treated sample
48:00:00 Time -20 °C Freezer
Note
The tissue disruption tube is a provided material in the QIAGEN DNeasy Plant Pro Kit
2d
Sample Disruption & Lysis
25m
After incubation, add 500 µl of Solution CD1 into the tissue disruption tube;
500 µL CD1
Note
As recommended by the manufacturer's instructions: If your sample is high in phenolic compounds, add 450 µl Solution CD1 and 50 µl Solution PS.
1m
Vortex to initial mix to disrupt the sample in the added solution;
3m
Parafilm the tube lid. Vortex at maximum speed for 5 – 20 min (the observed average in my case 15 min);00:15:00 Average time
Note
Note 1: Parafilm well the tube lid. This is a security detail to preserve the sample.
Note 2: The duration depends specially on the biological mass (if ≤100mg, period will be 3 – 10 min) and the fungi aspect (a thin cellular barrier plumb – for mushroom or tissue aspect – for mycelia) (even if ≤100mg, period will be 10 – 15 min).
Note 3: It is primordial to observe the process in terms of the biological resource.
15m
Centrifuge the tissue disruption tubes at 12 000 g for 2 min;
12000 x g, 00:02:00 , Room Temperature
Note
Relative Centrifugal Force (RCF) = g = 1.118 * 10-5 * r (cm) * (rpm)2
2m
Transfer exactly 400 μl of the supernatant to a new, clean and autoclaved 1.5 ml microcentrifuge tube;
400 µL Transfer exact volume from the surprenatant
Note
Store the used tissue disruption tube with its pellet at -20°C for maximum 3 days, if the operator estimated another extraction round for the same sample.
2m
Add 200 µl of the cold Solution CD2 (4°C) and vortex for 5 s;
200 µL CD2
4 °C CD2 Solution should be stocked in refrigerator
Note
Take out the CD2 solution just before use and return it to the refrigerator directly after use.
1m
Centrifuge at 12 000 g for 1 min at room temperature;
12000 x g, 00:01:00 , Room Temperature
Avoiding the pellet, transfer exactly 450μl of the supernatant to a new, clean and autoclaved 1.5 ml microcentrifuge tube.
450 µL Transfer exact volume from the surprenatant
Note
We tested collecting the entire volume. This reduced the ratios of extracted DNA. The best samples were collected by taking care not to disturb the tube after centrifugation and by recovering only the upper portion.
1m
Inhibitors Removal
1m
Add 500 µl of Buffer APP and vortex for 5 s.
500 µL APP Buffer
1m
gDNA Binding, Washing and Elution
20m 20s
Load 600 µl lysate onto an MB Spin Column;
600 µL Lysate Solution
30s
Centrifuge at 12 000 g for 1 min;
12000 x g, 00:01:00 , Room Temperature
1m
Discard the flow-through;
Repeat steps 12 & 13: Add the remaining lysate volume in the MB Spin Column and repeat the centrifugation;
12000 x g, 00:01:00 , Room Temperature
1m
Centrifuge at 12 000 for 1 min to ensure that all the lysate has passed through the MB spin column;
12000 x g, 00:01:00 , Room Temperature
1m
Place the MB spin column into a new, clean and autoclaved 2 ml collection tube;
1m
Add 650 µl of Buffer AW1 to the MB spin column;
650 µL AW1
40s
Centrifuge at 12 000 g for 1 min;
12000 x g, 00:01:00 , Room Temperature
1m
Discard the flow-through, tap the tube on a hygienic paper to ensure that all excess liquid is removed, and place the MB spin column back into the same 2 ml collection tube;
45s
Centrifuge at 12 000 for 1 min to ensure that all the Buffer AW1 volume has passed through the MB spin column;
12000 x g, 00:01:00 , Room Temperature
1m
Add 650 µl of Buffer AW2 to the MB spin column;
650 µL AW2
40s
Centrifuge at 12 000 g for 1 min;
12000 x g, 00:01:00 , Room Temperature
1m
Discard the flow-through, tap the tube on a hygienic paper to ensure that all excess liquid is removed, and place the MB spin column back into the same 2 ml collection tube;
45s
Centrifuge at 15 000 for 4 min to ensure that all the remaining volume has passed through the MB spin column;
15000 x g, 00:04:00 , Room Temperature
4m
Place the MB spin column into a new, clean and autoclaved 1.5 ml microcentrifuge tube;
Note
Cut the tube cap before centrifuging, especially if you are manipulating more than two tube.
1m
Add 15-50 µl of buffer EB to the center of the white filter membrane;
15-50 µL EB
Incubate for 1-2min of contact;
00:02:00 Incubation as maximum
Note
Note 1: For Illumina technologies, add 30-60 µl of buffer EB.
Note 2: For Nanopore Oxford Technology (ONT), add 10-20 µl of buffer EB.
Note 3: For an optimum yield, it is better to use between 15-30 µl of buffer EB in the case of mycelia culture and 10-20 µl of buffer EB in the case of mushroom.
2m
Centrifuge at 13 000 g for 2 min;
10003 x g, 00:02:00 , Room Temperature
Note
Store the MB spin column (next to its used tissue disruption tube) at -20°C, if the operator estimated another extraction round for the same variety.
2m
Transfer the obtained volume into new, clean and autoclaved 1.5 ml microcentrifuge tube;
gDNA
1m
The DNA is now ready for downstream quantification, qualification and sequencing analyses.
Nevertheless, gDNA should be store at -20°C. When needed, it should be adapted for 12-24h at 4°C before daily uses. If gDNA volume is important, aliquot it and store the stock at -80°C.
-20 °C In the case of near future use
-80 °C In the case of aliquot storage
Note
Before any use, the samples should be adapted to the temperature.
Safety information
The uncontrolled descrease of samples temperature, especially in the case of ONT downstream analyses, may be the source of mutation or bad conservation of gDNA which would bias the output sequencing results.
Note
For sequencing with Illumina technologies, samples should be diluted in DNase and RNase free water. In contrast, Oxford Nanopore Technology typically requires more concentrated levels (no problem with EB).
UltraPure™ DNase/RNase-Free Distilled WaterThermo FisherCatalog #10977015
gDNA Quality Assessment Workflow
1h 5m
Purification control by NanoDrop Spectrophotometer;
Blanc: The Kit Elution Buffer (1µl)
Sample: 1µl gDNA sample
Note: Well clean between samples and measurments with autoclaved ultrapure water and towel
Importance: To have an idea about the gDNA purity (260/230 & 260/280)
Equipment
Nanodrop 2000C
NAME
Thermo Scientific
BRAND
TSC-ND2000C
SKU
5m
Concentration control by Qubit Fluorometer;
Sd1: 10µl Sd1 + 1µl Reagent + 189µl Buffer
Sd2: 10µl Sd2 + 1µl Reagent + 189µl Buffer
Sample: 1µl Sample + 1µl Reagent + 198µl buffer
Incubation: 2min at ambient temperature
Note 1: Use BR, if the estimated concentration by Nanodrop is greater than or equal to 1000 ng/µl. Otherwise, use HS.
Importance: To determine the exact concentration of gDNA double strand.
Equipment
Invitrogen™ Qubit™ 3 Fluorometer
NAME
Accurately measures DNA, RNA, and protein using the highly sensitive fluorescence-based Qubit quantitation assays
TYPE
Invitrogen™ Q33216
BRAND
Q33216
SKU
LINK
Quality and Integrity Estimation by Gel Electrophoresis with 1Kb size estimator;
Gel Percentage: 2% Agarose Gel
Running Buffer: 1xTris Borate EDTA (TBE)
Run: 1h/100V
Importance: To have an idea about the intensity of the gDNA band, to estimate its size, and to have a look in the quality of gDNA (wether degraded or not)
1h
If possible, it is highly recomanded to exactly quantify and ensure quality controle by Fragment Analyser.
Importance: To determine the disribution of gDNA fragment size, especially in the case of ONT. The best scenario is to have a unimodal distribution with a representative High Weight fragment (≥10Kb)