| Product Description | This kit combines efficient guanidine isothiocyanate lysis technology with silicon matrix membrane purification technology to efficiently extract total RNA from animal cells and tissues. The starting sample usually has a maximum of 30 mg of tissue or 1 x 107 cells. This reagent kit can also recover partially purified RNA, RNA obtained from in vitro transcription and enzymatic reactions. This reagent kit can extract and purify high-quality RNA with a molecular weight greater than 200 bases, with almost no DNA residue. If RNA experiments are to be conducted that are highly sensitive to trace amounts of DNA, residual DNA can be digested and removed on a column using DNase I without RNase. The extracted RNA can be used for downstream experiments such as RT-PCR, Northern Blot, Dot Blot, etc.
R666020
| Component | 50 T | Storage | | R666020A | Buffer RL | 35 mL | RT | | R666020B | Buffer RW1 | 40 mL | RT | | R666020C | Buffer RW2 (concentrate) | 11 mL | RT | | R666020D | RNase-Free Water | 10 mL | RT | | R666020E | Spin Columns RM with Collection Tubes | 50 sets | RT | | R666020F | RNase-Free Centrifuge Tubes (1.5 mL) | 50 EA | RT |
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Self prepared reagents: β- Mercaptoethanol, anhydrous ethanol (newly opened or dedicated for RNA extraction).
Preparation and important precautions before the experiment
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.
2. The extracted samples should avoid repeated freeze-thaw cycles, otherwise it will affect the quantity and quality of RNA extraction.
3. Before use, please check if there is any crystallization or precipitation in the Buffer RL. It can be heated at 56 ℃ and re solved. Please add Buffer RL before use β- Mercaptoethanol, with a final concentration of 1%. Add 10 to 1ml Buffer RL μ L β- Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month.
4. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.
5. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.
6. If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with DNase I that does not contain RNase. Operation steps
1. Sample processing
1a organization: Grind the organization in liquid nitrogen. Add 600 to every 20-30 mg of tissue μ L Buffer RL (check if it is added before use) β- Mercaptoethanol), tissue sample less than 20 mg plus 350 μ Buffer RL. The sample volume shall not exceed one tenth of the buffer RL volume.
1b Single layer culture of cells: The cells are directly lysed or processed into cell suspensions in a culture bottle, centrifuged to obtain cell precipitates, and the supernatant is discarded. 600 is added every 6-10 cm2 of culture area μ Buffer RL, less than 6 cm2, add 350 μ Blow buffer RL several times to fully crack it.
1c cell suspension: Centrifuge at 12000 rpm (~13400 × g) for 1 minute to discard the supernatant and obtain cell precipitate. Add 600 cells every 5 × 106-1 × 107 cells μ Buffer RL, less than 5 × 106 cells added to 350 μ Blow buffer RL several times to fully crack it.
Attention:
1) Try to eliminate the cell culture medium as much as possible, as it may inhibit cell lysis and affect RNA production.
2) Try to fully suspend and lyse the cells, otherwise it will affect RNA production.
2. After the sample is fully lysed, it should be left at room temperature for 5 minutes to completely separate the protein nucleic acid complex.
3. Centrifuge at 2000rpm for 2-5 minutes, take the supernatant and proceed to the next step.
4. Add 1 volume (600) μ L or 350 μ l) Mix 70% ethanol (prepared without RNase water) well.
Attention: Adding ethanol may cause precipitation and will not affect subsequent experiments.
5. Add all the solution obtained in step 4 to the Spin Columns RM that has 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 for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube. Attention: The maximum loading capacity of the adsorption column is 100 µ g, do not overload, otherwise it will affect the yield and purity of RNA.
6. Add 700 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 in the collection tube.
Optional steps: If conducting RNA experiments that are highly sensitive to trace amounts of DNA, replace step 6 with the following steps.
1) Add 350 to the adsorption column μ L Buffer RW1, centrifuge at 12000 rpm for 15 seconds, discard the waste liquid, and place the adsorption column back into the recovery manifold.
2) Preparation of DNase I mixture: Take 52 μ Add 8 RNase Free Water to it μ 10 x Reaction Buffer and 20 μ DNase I (1 U/ μ l) Mix well and prepare to a final volume of 80 μ The reaction solution of L.
Attention: The above system is configured according to our company's DNase I reaction system. Please refer to the corresponding manual for other company products. 3) Add 80 µ l of the prepared DNase I reaction solution directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.
4) Add 350 to the adsorption column μ L Buffer RW1, centrifuge at 12000 rpm for 15 seconds, discard the waste liquid, and place the adsorption column back into the recovery manifold.
7. 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 in the collection tube.
8. Repeat step 7.
9.
Centrifuge at 12000 rpm for 2 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.
Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).
10. Place the adsorption column in a new RNase free centrifuge tube, and add 30-50 to the middle of the adsorption column in the air μ Place RNase Free Water at room temperature for 1 minute, 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 30 μ l. Small volume affects the recovery rate.
2) If you want to increase RNA production, you can use 30-50 μ Repeat step 10 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 10.
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