Preparation of fungal total RNA
Preparation of fungal total RNA
Total RNA was extracted from a variety of different fungi.
Operation method
Preparation of fungal total RNA
Materials and Instruments
Phenol Chloroform Isoamyl alcohol 12mol L Lithium chloride 3mol L Sodium acetate Ethanol DEPC treated water Move I. Materials and equipment Caveat 1) When transferring ground fungi into Falcon tubes, do so carefully to avoid loss. All operations should be done quickly and samples/tubes should be kept on ice to minimize the effect of RMase.2) Heat at 65°C to help dissolve the RNA. If there are insoluble particles, centrifuge at 13,000 to 18,000 g for 2 min and collect the supernatant, which contains some or very little RNA or no RNA. For more product details, please visit Aladdin Scientific website.
Water bath Low-temperature high-speed centrifuge
1) Phenol: chloroform: isoamyl alcohol (25:24:1).
2) Chloroform: Isoamyl alcohol (2:1)
3) 12mol/L lithium chloride.
4) 3mol/L sodium acetate.
5)70% ethanol.
6)DEPC treated water
7) Water bath.
8) Cryogenic high-speed centrifuge.
II Methods of operation
1) Grind 2~5 g of fungal tissue in a mortar with liquid nitrogen until it is completely powdered.
2) Transfer the ground powder to a Falcon tube containing extraction buffer (3 mL of extraction buffer per gram of fungus) and an equal volume of phenol: chloroform: isoamyl alcohol (preheated to 65°C).
3) Shake the tube to mix thoroughly.
4) Transfer the mixture to an RNase-free centrifuge tube and centrifuge at 13,000~18,000 g for 30 min at 4℃.
5) Collect the upper aqueous phase, add an equal volume of chloroform:isoamyl alcohol (24:1) and mix by turning.
6) Centrifuge at 1300~18000 g for 30 min at 4℃.
7) Collect the upper aqueous phase and add 12mol/L lithium chloride to a final concentration higher than 2mol/L. The final concentration of the aqueous phase was then increased to 2mol/L.
8) Mix and leave at 4°C overnight to precipitate RNA.
9) Centrifuge at 13000~18000 g for 30 min at 4℃.
10) remove the supernatant, wash the precipitate with 5 ml of 3mol/L sodium acetate (pH5.2), and centrifuge for 15 min as in step 9).
11) Remove supernatant. Wash the precipitate with 20 ml of 70% ethanol and centrifuge for 15 min as in step 9).
12) Repeat with 70% ethanol.
13) Vacuum dry to remove ethanol.
14) Dissolve the precipitate in an appropriate volume (250~lOOOul) of DEPC-treated water.
15) Store the dissolved RNA at -70°C.