Fungal RNA extraction can be: (1) used in the study of fungal RNA interference mechanism; (2) used in cDNA library construction and other studies; (3) used in the corresponding study of fungal molecular biology.
Operation method
Liquid Nitrogen Grinding
Principle
Liquid nitrogen milling lyses the cells, inducing dissociation of the nucleoprotein bodies, separating RNA from proteins, and releasing the RNA into solution. When chloroform is added, it abstracts acidic phenol, which drives RNA into the aqueous phase, and centrifugation creates an aqueous and an organic layer so that the RNA separates from the proteins and DNA that remain in the organic phase.
Materials and Instruments
Mycelium Move 1. Preheat the RNA extract in a water bath at 65°C before the start of the experiment, and add ME (mercaptoethanol) to the centrifuge tubes, (80 ul in 10 mL and 300 ul in 50 mL). Caveat Extract RNA as low as possible, and if conditions do not permit, operate at room temperature for as short a time as possible. Common Problems Experimental reagents preparation 1. RNA extraction buffer (CTAB): 2% CTAB (W/V), 2% polyvinylpyrrolidone PVP (W/V), 100 mM Tris-HCl (pH 8.0, DEPC-treated water configuration), 25 mM EDTA, 0.5 g/L spermidine Spermidine, 2.0 M NaCl, 2% mercaptoethanol (V/V, added before use). added before use). Since Tris-HCl reacts with DEPC under autoclaving conditions, RNA extraction buffer can be prepared directly with DEPC-treated water. 2. SSTE: 1 M NaCl, 0.5% SDS (W/V), 10 mM Tris-HCl pH 8.0, 1.0 mM EDTA. 3. 10 M LiCl. 3. 10 M LiCl Directly prepare 10 M LiCl with distilled water, add 1‰ DEPC overnight and autoclave. 4. DEPC treated water Treat distilled water with 1‰ DEPC overnight and autoclave. 5. 3 M NaAc, chloroform:isoamyl alcohol (24:1), phenol (pH 4.5):chloroform:isoamyl alcohol (25:24:1), anhydrous ethanol, 70% alcohol. For more product details, please visit Aladdin Scientific website.
NaCl Distilled water SDS Tris-HCl EDTA DEPC SSTE Ethanol Alcohol Phenol: Chloroform: Isoamyl alcohol LiCl
Liquid Nitrogen Tank Centrifuge Tube Centrifuge Water Bath Refrigerator UV Spectrophotometer Electrophoresis Instrument
2. Take about 0.8 g of mycelium (mycelium obtained from liquid culture can be filtered by vacuum extraction, solid culture is better to say), quickly grind it into fine powder in liquid nitrogen, load it into a 50 mL centrifuge tube, add the preheated RNA extraction solution in the amount of 8 mL of 1 g of material, and mix it by inverting.
3. 65℃ water bath for 3-10 min, mixing 2-3 times during the period.
4. Add equal volumes of phenol (note acid phenol pH 4.5):chloroform:isoamyl alcohol (25:24:1) extraction (10 000 rpm, 4°C, 5 min).
5. Take the supernatant and extract with an equal volume of chloroform:isoamyl alcohol (24:1) (10 000 rpm, 4°C, 5 min).
6. Add 1/4V volume of 10 M LiCl solution and leave at 4°C for more than 6h (or overnight).
7. Centrifuge at 10 000 rpm, 4°C for 20 min.
8. Discard supernatant and dissolve precipitate with 500 ul SSTE.
9. Phenol:chloroform:isoamyl alcohol (25:24:1) was extracted twice and chloroform:isoamyl alcohol (24:1) was extracted once (10 000 rpm, 4°C, 5 min).
10. Add 2V volume of anhydrous ethanol and precipitate for more than 30 min at -70°C in a refrigerator.
11. Centrifuge at 12 000 rpm, 4°C for 20 min.
12. Discard the supernatant. The precipitate was rinsed once with 70% alcohol and dried.
13. Add 200 ul of DEPC-treated water to dissolve.
14. Detect the quality of RNA by non-denaturing agarose gel electrophoresis and UV spectrophotometer scanning.(During the extraction process, if the protein content or other impurities are still high, the number of extractions can be increased.)