1. Concept The process of RNA virus replication will produce dsRNA with a length similar to the genome, and the progeny genome of some dsRNA viruses is also dsRNA, and these dsRNA are much more stable than ssRNA. Therefore, RNA viruses can be identified and analyzed by simple dsRNA analysis technology. The dsRNA extraction technology was developed using the principle that CF-11 cellulose can specifically adsorb dsRNA at a certain alcohol concentration. The purified dsRNA can be used as a template after denaturation to synthesize cDNA or perform RT-PCR amplification. Add artificial DNA linkers at both ends of the dsRNA to connect the dsRNA and DNA under the action of ligase, clone and sequence them to obtain the unknown virus sequence. 2. Materials and utensils Cellulose (CF-11); disposable plastic syringe as; mortar; 50m1 centrifuge tube; Liquid nitrogen; mercaptoethanol; saturated phenol; chloroform; SDS; absolute ethanol; isopropanol; agarose; Tris base; EDTA; NaCl. 3. Test procedure 1. Take 15g of freshly veined leaf tissue and grind it into powder in liquid nitrogen. 2. Add 10m1 2 * STE (PH8.0), 300ul mercaptoethanol, 6m1 saturated phenol, 4m1 chloroform, 1.4ml 10% SDS, and homogenize for 20min. 3. Centrifuge at 15000g at 4 ° C for 15 minutes, and collect the supernatant. 4. The supernatant was adjusted to 17% ethanol-containing RNA mixture with absolute ethanol and mixed at room temperature. 5. Weigh 3g of cellulose (CF-11), equilibrate with 1 * STE containing 17% ethanol and then install the column, using a 51ml disposable plastic syringe as the chromatography column. 6. Load the RNA mixture and wash the column with 90ml of 1 * STE containing 17% ethanol. 7. Elute with 1 * STE without ethanol, discard the first 5ml, collect the next 10-15ml, add an equal volume of isopropanol, mix and place at room temperature for 5min. 8. Centrifuge at 15000g for 15 minutes at 4 ° C, and discard the supernatant. 9. Precipitate with 200ul 70% ethanol and vacuum dry for 3min. 1 10. Dissolve the precipitate in 60ul TE in time and store at -20 ℃. 11. Take 5ul of solution mixed with 1% agarose gel electrophoresis analysis, compare the relative size of dsRNA, and recover the required bands if necessary. 12. According to the size of the dsRNA displayed by gel electrophoresis, compare it with the known RNA virus dsRNA or relative DNA molecular weight marker to determine the size of the virus genome RNA species to be tested. Experience the new blog
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Plant virus double-stranded RNA (dsRNA) extraction