Extraction of the total RNA of fungal mycelium

:
RNA Extraction Buffer (CTAB): 2% CTAB (W/V), 2% Polyethylene PVP (W/V), 100 mM Tris-HCl (pH8.0, DEPC-treated water configuration), 25mM
EDTA
, 0.5g/L spermidine, 2.0M NaCl, 2%ethanol (V/V, added before use). Since Tris-HCl reacts with DEPC under high temperature sterilization conditions, water preparation treated directly with DEPC when the buffer is extracted with RNA.
SSTE：1 M NaCl,0.5%
SDS
(W/V), 10 mM Tris-HCl pH8.0, 1.0 mM EDTA
10M LiCl is directly sterilized at high temperatures after overnight with distilled water with 10M LiCl, plus 1 inDEPC.
3M NaAc
chloroform: isoquinol (24:1)
phenol (pH4.5): chloroform: isoquinol (25:24:1)
DEPC treatment water distilled water overnight with a DEPC treatment of 1 1 s/ 1, high temperature sterilization
waterless ethanol
70% alcoholmethod:
1. The RNA extract was preheated in a 65-degree C water bath before the experiment began, and ME (-based ethanol) was added to the centrifugal tube (10mL plus 80ul, 50mL with 300ul).
2. Take about 0.8g mycelium (liquid
culture
obtained by vacuum filtering can be! Solid culture is better said), in liquid nitrogen quickly ground into a fine powder, loaded into a 50 mL centrifuge tube, according to the amount of 1g material 8mL to add preheated RNA extract, mixed upside down.
3. Water bath 3-10 min at 65 degrees C, mixing 2-3 times during
4. Add iso-volume phenols (note acid pH4.5): chloroform: isoquinol (25:24:1) withdrawal (10,000 rpm, 4 degrees C, 5 min)
5. Take clear, equal volume chloroform: isosterol (24:1) withdrawal (10,000 rpm, 4 degrees C, 5 min)
6. Add 1/4V volume 10M LiCl solution, place more than 6hr (or overnight) at 4 degrees C
7. 10,000 rpm, 4 degrees C centrifugation 20 min
8. Discarded, dissolved precipitation with 500 ulSSTE
9. Phenols: chloroform: isotrol (25:24:1) pumped twice, chloroform: isosterol (24:1) pumped 1 time (10,000) rpm, 4 degrees C, 5 min)
10. Plus 2V volume of waterless ethanol, precipitation above 30 min in a refrigerator at -70 degrees C
11. 12,000 rpm, 4 degrees C centrifugation 20 min
12. Discard the Qing. Precipitation is rinsed once with 70% alcohol,
dry
13. Plus 200 ul dePC treatment water dissolved
14. Using non-denatured
agar
sugar gel electrophoresis and ultraviolet
phosphorescometer
scan to detect the mass of RNA
(in the extraction process, if the
protein
content or other impurities are more, can increase the number of withdrawals