| Product Description | This kit is used to extract and purify high-quality total RNA from various plants, and is also suitable for the extraction of fungal hyphal RNA. A unique Shredder separation column is used for homogenization and filtration of high viscosity plant or fungal lysates, while silica based membrane is used to adsorb RNA for purification, effectively removing various pollutants such as polysaccharides through washing. The washed RNA can be directly used in various downstream experiments. RNA with a molecular weight greater than 200 bases was extracted using this reagent kit, with high purity and almost no DNA residue. If it is an RNA experiment that is very sensitive to trace amounts of DNA, the remaining DNA can be digested and removed on a column using DNase I without RNase. The extracted RNA can be used for experiments such as Northern Blot, Dot Blot, RT-PCR, and in vitro translation.
| R665489 | Component | 50 T | Storage | | R665489A | Buffer RL | 35 mL | RT | | R665489B | Buffer RLC | 35 mL | RT | | R665489C | Buffer RW1 | 40 mL | RT | | R665489D | Buffer RW2 (concentrate) | 11 mL | RT | | R665489E | RNase-Free Water | 10 mL | RT | | R665489F | Spin Columns FL with Collection Tubes | 50 sets | RT | | R665489G | Spin Columns RM with Collection Tubes | 50 sets | RT | | R665489H | 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. Please add Buffer RL before use β- Mercaptoethanol, with a final concentration of 1%. Add 10 to 1 ml Buffer RL μ L β Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month. No need to add buffer RLC when using it β- Mercaptoethanol.
Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.
5. If precipitation occurs in Buffer RL and Buffer RLC, please heat them to dissolve and place them 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.
7. If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with DNase I without RNase. Operation steps:
1. Take 50-100 mg of fresh plant tissue, add liquid nitrogen and quickly grind it into powder.
2. Collect the ground powder into a centrifuge tube (provided by oneself) and add 600 μ L Buffer RL (check if it is added before use) β- Sulfhydryl ethanol or Buffer RLC, vortex oscillation causes it to fully decompose.
Attention:
1) The main component of Buffer RL is guanidine isothiocyanate, which is suitable for the lysis of most plant tissues. However, in some plant tissues (such as corn endosperm), due to the unique secondary metabolites, guanidine isothiocyanate causes precipitation in the sample, resulting in poor RNA extraction efficiency. In this case, Buffer RLC can be added instead of Buffer RL.
2) Incubating at 56 ℃ for 1-3 minutes helps with tissue lysis, but plants with high starch content should not be subjected to high-temperature incubation.
3. Transfer all the liquid obtained in step 2 to the spin columns FL that have been loaded into the collection tube, centrifuge at 12000 rpm (~13400 × g) for 2 minutes, and transfer the supernatant from the collection tube to a new centrifuge tube (provided by oneself).
Attention:
1) When aspirating liquid, the tip of the gun can be cut off for easy sampling.
2) Spin Columns FL can remove most of the fragments, but there will still be a small amount flowing out. After centrifugation, precipitation will form in the collection tube. When proceeding to the next step, be careful not to absorb the sediment.
4. Add 0.5 times the volume of anhydrous ethanol to the clean cracking solution obtained in step 3 and quickly mix well. Attention: Adding ethanol may cause precipitation, but it does not affect subsequent experiments.
5. Add all the solutions obtained in step 4 to the spin columns RM that have been loaded into the collection tube. If it is not possible to add all the solutions to the adsorption column at once, please transfer them in two separate steps. Centrifuge at 12000 rpm for 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
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 back into 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 instructions for other company products.
3) Add 80 µ l of 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 15 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
8. Repeat step 7.
Centrifuge at 9.12000 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 dry the anhydrous ethanol in the column.
Attention:
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.).
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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