Apr 02, 2025
DNA Extraction from Magnaporthe oryzae (PCR quality)
- Sophia Haeussler1,
- Ulrich Lutz2,
- Thorsten Langner1
- 1Max-Planck-Institute for Biology, 72076 Tübingen;
- 2Biogenda, 72076 Tübingen
- Magnaporthe Protocols
Protocol Citation: Sophia Haeussler, Ulrich Lutz, Thorsten Langner 2025. DNA Extraction from Magnaporthe oryzae (PCR quality). protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9rzd4v3e/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: January 21, 2025
Last Modified: April 02, 2025
Protocol Integer ID: 118794
Funders Acknowledgements:
European Research Council (ERC-2022-Stg)
Grant ID: 101077853
Abstract
Genomic DNA extractions are crucial for genetic manipulation, confirmation of transformants, or molecular surveillance. Often, extraction of good-quality genomic DNA for PCR-amplification is required. Here we provide a protocol for DNA extraction from fungal tissue. The protocol was tested for Magnaporthe oryzae and the extracted DNA used for PCR. A yield of 4 – 20 µg of DNA per sample with A260/A280 ratio of 2.1 and A260/A230 ratio of 2.2 can be expected.
Guidelines
The protocol was tested for M. oryzae DNA extractions. For PCR, the concentration was adjusted to 60 ng/µl and 1 µl used in a 20 µl PCR reaction.
Materials
Reagents
Extraction buffer
- 8 M Guanidine Hydrochloride (15.3 g for 20 ml)
- 20 mM MES (0.078 g for 20 ml)
- 20 mM EDTA (800 µl 0.5 M EDTA, pH = 8.0 for 20 ml)
Add MilliQ water to 20 ml, autoclave.
Add 100 µl RNAse A per 20 ml buffer directly before starting DNA extraction.
96 % Ethanol
70 % Ethanol
MilliQ water (for elution)
- 50 ml 20X Nitrate Salts (See Below)
- 1 ml Trace Elements (See Below)
- 10 g D-Glucose
- 2 g Peptone
- 1 g Yeast Extract
- 1 g Casamino Acids
- 1 ml Vitamin Solution (See Below)
ddH2O to 1 l
Adjust pH to 6.5 with NaOH
Autoclave
20 X Nitrate Salts
- 120 g NaNO3
- 10.4 g KCl
- 10.4 g MgSO4*7H2O (5.2 g if anhydrous)
- 30.4 g KH2PO4
ddHOH to 1 L
Autoclave
Store at 4°C.
1000 X Trace Elements
Add the compounds in order!
- 80 ml ddH2O
- 2.2 g ZnSO4*7H2O
- 1.1 g H3BO3
- 0.5 g MnCl2*4H2O
- 0.5 g FeSO4*7H2O
- 0.17 g CoCl2*6H2O
- 0.16 g CuSO4*5H2O
- 0.15 g Na2MoO4*2H2O
- 5 g Na4EDTA
Boil briefly and let cool to 60°C
Adjust pH to 6.5 with KOH
Cool to room temperature
ddH2O to 100 ml
Store at 4°C
1000x Vitamin Solution
- 0.01 g Biotin
- 0.01 g Pyridoxin
- 0.01 g Thiamine
- 0.01 g Riboflavin
- 0.01 g PABA (p-aminobenzoic acid)
- 0.01 g Nicotinic Acid
- 100 ml ddH2O
Store in a dark glass bottle at 4°C
Ethanol (70% for sterilization)
Liquid nitrogen (for grinding the sample tissue)
Materials
- Miracloth
- Mortar and pestle
- Econospin columns (1920-250, EpoChlifescience) or AHN myTube SC 0.8 mL filter tube, GF-F2 filter, 2.0 mL receiver tubes
- Steel spatula and steel forceps
Fungal growth in liquid culture
Fungal growth in liquid culture
Steps 2-6 are performed in a sterile hood.
Prepare a M. oryzae culture by placing a filter paper stock on complete-medium agar and let it grow for 7 days at 25 °C.
Place a forceps and a small (5 mm width) spatula into Ethanol, flame-sterilise and let cool down.
Cut out 6-8 agar plugs (5 mm x 5 mm) from the outer edge of the colony (i.e., young vegetative hyphae) using the sterilized spatula.
Pour 50 ml of sterile complete medium into a sterilized 100 ml erlenmeyer flask.
Transfer the agar plugs into the liquid complete medium using sterilized forceps or a sterilized spatula.
Incubate the liquid culture on a rotary shaker at 25 °C and 120 rpm for 2 days.
Note
During the incubation, the mycelium grows out of the agar blocks and forms small balls. The medium remains transparent. If the medium gets milky, it likely indicates a bacterial contamination of the medium.
Tissue harvesting and ginding
Tissue harvesting and ginding
Filter the liquid culture through 2 layers of Miracloth. Wash the mycelium with sterile MilliQ water.
Squeeze the miracloth to dry the culture as much as possible.
Note
If there is liquid remaining in the harvested mycelium, place between two watman filter papers and squeeze out the remaining liquid.
Pre-cool a mortar and pestle, a small spatula and a 1.5 ml reaction tube in liquid nitrogen. Place the mycelium in a mortar and add liquid nitrogen.
Grind the mycelium to a fine white powder.
Note
Prevent the mycelium/powder from thawing. If necessary, add more liquid nitrogen to cool down the mortar again.
Using the pre-cooled spatula, transfer the ground mycelium powder into the precooled 1.5 ml reaction tube up to the 100 µl mark. Close the lid and freeze the tube in liquid nitrogen. Proceed with DNA extraction immediately or store the tissue at -20 °C for storage until the next step.
DNA extraction with Econospin columns
DNA extraction with Econospin columns
Preheat a water bath to 60 °C. Add RNAse A (3 µl/600 µl volume) to extraction buffer.
Add 600 µl extraction buffer (with RNAse A) to each tube. Vortex thoroughly.
Place tubes in 60 °C water bath for at least 10 min.
Centrifuge at 20.000 rcf for 1 min.
Transfer 400 µl of clear supernatant to a new 1.5 ml tube.
Add 1x volume of 96 % Ethanol (400 µl) and mix by inverting 10 times.
Transfer 800 µl to an Econospin column (1920-250, EpoChlifescience).
Note
AHN columns can be used alternatively (AHN myTube SC 0.8 mL filter tube, GF-F2 filter, 2.0 mL receiver tube).
Centrifuge at 20.000 rcf for 1 min. Discard flow-through.
Add 450 µl 70 % Ethanol. Centrifuge at 20.000 rcf for 1 min.
Add 350 µl 70 % Ethanol. Centrifuge at 20.000 rcf for 1 min. Discard flow-through.
Place the Econospin column back into the collection tube and dry spin at 20.000 rcf for 2 min.
Place Econospin column into a new 1.5 ml tube. Add 40 µl MilliQ water (optional: preheated to 55 °C). Incubate for 1 min. Centrifuge at 20.000 rcf for 1 min.
Note
Preheating the water before elution can increase the yield.