Experimental Step 1. Extract total RNA using RNeasy Plant Mini Kit
1) Add 0.5 ml RLT buffer and 5ul β-mercaptoethanol to a 1.5 ml centrifuge tube;
2) Weigh about 100 mg of material, transfer it to the extract after grinding with liquid nitrogen, vortex to mix, 56 ° C, 2 min;
3) Add a purple column, centrifuge at 23 ° C, 12000 rpm for 2 min;
4) Transfer the filtrate to a new 1.5 ml centrifuge tube, add 1/2 volume of absolute ethanol, mix with a pipette tip, transfer to a pink column, 23 ℃, 12000 rpm, centrifuge for 15 sec;
5) Discard the filtrate, add 700 ul RW 1, 23 ℃, 12000 rpm, centrifuge for 15 sec;
6) Move the column into a new 2 ml collection tube, add 500 ul RPE, 23 ℃, 12000 rpm, centrifuge for 15 sec;
7) Discard the filtrate, add 500ul RPE, 23 ℃, 12000 rpm, centrifuge for 2 min;
8) Add 20 ul of DEPC treated water, let stand at room temperature for 15 min, 23 ℃, 12000 rpm, centrifuge for 1 min;
9) Add 40 ul and 20 ul of DEPC-treated water, let stand at room temperature for 15 min, centrifuge at 23 ° C, 12000 rpm for 1 min;
10) Combine the three collected RNAs and take 2ul of electrophoresis to check the purity and quality. -20 ℃ short-term storage, or -80 ℃ long-term storage.
2. Extract RNA using TRIZOL kit
1) Add 1 ml of TRIZOL extract to a 1.5 ml centrifuge tube;
2) Weigh about 100 mg of liquid nitrogen milled material, transfer to the extraction solution, mix well, centrifuge at 2-8 ° C, ≤12000 g for 10-15 min;
3) Take the supernatant, put it at 15-30 ° C for 5min, add 0.2 ml of chloroform, shake vigorously for 15 sec, put it at 15-30 ° C for 2-3min, 2-80C, ≤12000 g, and centrifuge for 15 min;
4) Take the water phase, add an equal volume of isopropanol, place at 15-30 ℃ for 10 min, 2-8 ℃, <12000 g, and centrifuge for 10 min;
5) Wash the precipitate with 75% ethanol, centrifuge at 2-8 ° C, <7500g for 5 min; dissolve the RNA in DEPC-treated water, and store it at -20 ° C for a short time or -80 ° C for a long time
3. CTAB method to extract total plant RNA
1) Add 15 ml CTAB extraction buffer (need to add 300ul β-mercaptoethanol) to 50 ml centrifuge tube, preheat at 65 ℃;
2) Weigh 2-3 g of fresh plant material and grind it into powder in liquid nitrogen;
3) Transfer the sample to a 50 ml centrifuge tube, shake and mix quickly until there is no unity block, warm bath at 65 ° C for 10 min, then add 15 ml of chloroform, and mix;
4) At room temperature, centrifuge at 10,000 rpm for 15 min, the upper aqueous phase is transferred into a new centrifuge tube, add equal volume of chloroform, mix again and extract again;
5) Transfer the upper aqueous phase to another centrifuge tube, add 8M LiCl to make the final concentration 2M (approximately 1/3 of the aqueous phase added), 4 ℃, overnight;
6) 4 ℃, 12000 rpm, centrifuge for 20 min to precipitate RNA, discard the supernatant, wash the precipitate with 70% ethanol, and dry it;
7) Add 500ul SSTE to dissolve the precipitate, transfer to a 1.5 ml centrifuge tube, add an equal volume of acidic phenol / chloroform and mix well;
8) At room temperature, centrifuge at 13000 rpm for 10 min, transfer the upper aqueous phase to another 1.5 ml centrifuge tube, add an equal volume of chloroform and mix well;
9) At room temperature, centrifuge at 13000 rpm for 10 min, transfer the upper aqueous phase to a new tube, add 1/10 volume of 3M NaAC and 2 volumes of absolute ethanol, and let stand at -80 ° C for 30 min;
10) 4 ℃, 13000 rpm, centrifugation for 20 min, 70% ethanol to wash the precipitate. After drying, add 50 ul of DEPC to treat H20 to dissolve RNA, take 1 ul of electrophoresis, and store at -80 ℃ for later use.
4. RNA purification
1) In a 1.5 ml centrifuge tube treated with DEPC water, add: 45ul RNA, 7.5ul l0XDnase Assay Buffer, 20U RNase inhibitor 1.5ul Dnase I, and the mixture is incubated at 37 ° C for 30 min;
2) Adjust the volume of the solution to 250ul with DEPC-H20, add 1/10 volume of 3 M NaAC (pH 5.2) and 2 volumes of cold absolute ethanol, mix well, and place at -20 ℃ for 30 min;
3) 4 ℃, 12000 rpm, centrifugation for 20 min, RNA precipitation;
4) Wash once with 70% ethanol pre-cooled at -20 ℃ and once with 100% ethanol pre-cooled at -20 ℃;
5) After drying, dissolve the H2O treated with 20 ul DEPC and store at -20 ℃.
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