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Fungal DNA and RNA Extraction Methods
The study of fungal gene function often requires the extraction of DNA and RNA. However, due to the high content of polysaccharides in fungi, it can be challenging to obtain high-quality nucleic acids for downstream analysis. Therefore, finding an efficient and cost-effective method is essential. Below, I share a reliable method that I have been using successfully over the years.
Fungal DNA Extraction (Method 1)
1. Take 0.5g of fungal mycelium and quickly grind it into a fine powder using liquid nitrogen.
2. Add 4mL of extraction buffer and mix thoroughly by shaking.
3. Add an equal volume of isoamyl alcohol (24:1 ratio) and vortex for 3–5 minutes. This step avoids the use of phenol, saving both time and cost.
4. Centrifuge at 1000 rpm for 5 minutes at 4°C.
5. Transfer the supernatant to a new tube and re-extract with isoamyl alcohol (24:1). Centrifuge again at 10,000 rpm for 5 minutes at 4°C.
6. Add 2/3 volume of pre-cooled isopropanol (-20°C) or 2.5 volumes of absolute ethanol. Mix well and let stand for about 30 minutes.
7. Use a capillary rod to collect the precipitate. Rinse it with 75% ethanol, then with absolute ethanol. Dry gently and resuspend in 500μL of TE buffer.
8. Add 1μL of RNase A (10 mg/mL) and incubate at 37°C for 1 hour to remove any residual RNA.
9. Perform one more extraction with phenol (pH 8.0): isoamyl alcohol (25:24:1) and isoamyl alcohol (24:1). Centrifuge at 10,000 rpm for 5 minutes at 4°C.
10. Take the supernatant, add 1/10 volume of 3M NaAc and 2.5 volumes of absolute ethanol. Precipitate at -70°C for at least 30 minutes.
11. Wash the pellet with 75% ethanol, air dry, and dissolve in 200μL of TE buffer. Store at -20°C for long-term use.
This method ensures high-quality DNA suitable for PCR, sequencing, and other molecular techniques. It also minimizes the use of expensive reagents while maintaining efficiency and purity.
For RNA extraction, similar principles apply, but additional steps are required to prevent RNA degradation. Using a dedicated RNA isolation kit or modifying this protocol with a glycogen carrier can improve yield and quality. Always work in a clean environment and use RNase-free equipment to avoid contamination.
Whether you're working on fungal genomics, transcriptomics, or functional studies, having a solid extraction protocol is crucial. These methods can be adapted based on the specific fungal species and experimental needs.
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