| Product Description | This reagent kit uses an adsorption column that can specifically bind to viral RNA and a unique buffer system, suitable for isolating viral RNA from cell-free body fluids such as serum, plasma, urine, cerebrospinal fluid, and cell culture supernatants. The viral RNA specifically binds to the silicon substrate membrane, and pollutants flow through the membrane. Completely remove impurities such as proteins through two efficient washes, and then wash high-purity viral RNA with RNase free water or RNase Free Water provided by the reagent kit. The virus RNA extracted by this kit can be directly used for experiments such as RT-PCR, Real time RT-PCR, and Western blot analysis.
| R666005 | Component | 50 T | Storage | | R666005A | Buffer GL | 15 mL | RT | | R666005B | Buffer RW1 | 40 mL | RT | | R666005C | Buffer RW2（concentrate） | 11 mL | RT | | R666005D | Proteinase K | 12.5 mg | RT | | R666005E | Proteinase K Storage Buffer | 1.25 mL | RT | | R666005F | RNase-Free Water | 10 mL | RT | | R666005G | Spin Columns RS with Collection Tubes | 50 sets | RT | | R666005H | RNase-Free Centrifuge Tubes（1.5 mL） | 50 EA | RT |
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Self prepared reagent: anhydrous ethanol, 0.9% NaCl.
Preparation and important precautions before the experiment
1. Add 1.25 ml of Protein K Storage Buffer to Protein K to dissolve it and store at -20 ℃. The prepared Protein K should not be left at room temperature for a long time to avoid repeated freeze-thaw cycles, which may affect its activity.
2. To prevent RNase pollution, attention should be paid to the following aspects:
1) Use RNase free plastic products and gun heads to avoid cross contamination.
2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.
3) Prepare the solution using water without RNase.
4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.
3. Serum or plasma should avoid repeated freeze-thaw cycles that may cause protein denaturation or precipitation, reduce viral titers, and thus affect the yield of extracted viral nucleic acids.
4. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.
5. If buffer GL precipitates, it can be heated at 56 ℃ to dissolve and then placed at room temperature.
6. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly. Operation steps
1. Take 200 at room temperature μ Add serum or plasma to a 1.5 ml centrifuge tube (self provided). Attention: Less than 200 μ 0.9% NaCl (provided by the customer) can be added to make up for it.
2. Add 20 to the solution in the previous step μ Protein K, mix well.
3. Add 200 μ L Buffer GL, vortex oscillation for 15 seconds. Note: Do not directly add Protein K to Buffer GL.
4.
Incubate at 56 ℃ for 15 minutes, briefly centrifuge, and collect the solution on the tube wall to the bottom of the tube.
5. Add 250 μ Anhydrous ethanol, vortex for 15 seconds, incubate at room temperature for 5 minutes, briefly centrifuge, and collect the solution from the tube wall to the bottom of the tube.
6. Add all the solution obtained in step 5 to the Spin Columns RS that have been loaded into the collection tube. If it is not possible to add all the solution to the adsorption column at once, please transfer it in two batches, centrifuge at 12000 rpm (~13400 × g) for 1 minute, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
7. Add 500 to the adsorption column μ Centrifuge at 12000 rpm for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.
8. Add 500 to the adsorption column μ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
9. Add 500 to the adsorption column μ Centrifuge anhydrous ethanol at 12000 rpm for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column back into the collection tube.
10.
Centrifuge at 12000 rpm for 3 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry. Attention:
1) The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).
2) Recommended steps: Place the adsorption column into a new 1.5 ml centrifuge tube (provided), open the tube cover, and incubate in a 56 ℃ oven for 3 minutes to thoroughly dry the membrane of the adsorption column.
11. Place the adsorption column in a new RNase free centrifuge tube and add 20-50 to the middle of the adsorption column in the air μ Place RNase Free Water at room temperature for 5 minutes, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -70 ℃ to prevent degradation.
Attention:
1) The volume of RNase Free Water should not be less than 20 μ l. Small volume affects the recovery rate.
2) If you want to increase RNA production, you can use 20-50 μ Repeat step 11 for the new RNase Free Water.
3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 11.
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