| Product Description |
Product content | U670000 | Component | 50 T | 200 T | Storage | | U670000A | TRIzon Reagent | 60 mL | 2×110 mL | 2-8℃. Protect from light
| | U670000B | TRIzon PaI ™ | 10 mL | 2x20 mL | 2-8℃. Protect from light
| | U670000C | Buffer RW1 | 40 mL | 160 mL | RT | | U670000D | Buffer RW2 (concentrate) | 11 mL | 50 mL | RT | | U670000E | RNase-Free Water | 10 mL | 50 mL | RT | | U670000F | Spin Columns RM with Collection Tubes | 50 EA | 200 EA | RT | | U670000G | RNase-Free Centrifuge Tubes (1.5 mL) | 50 EA | 200 EA | RT |
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Product Introduction This kit is an improved column total RNA extraction kit based on TRIzon. The lysate is fully lysed and homogenized, and a unique silicon matrix membrane adsorption technology is used to efficiently and specifically bind RNA in a high salt state through a centrifugal adsorption column. At the same time, proteins, inorganic salt ions, and organic impurities are effectively removed to the maximum extent possible; Total RNA can be quickly extracted from animal tissues, plant materials, various microorganisms, and cultured cells. It can process 30-50 mg of tissue or 5 × 10 ⁶ cells each time, and can simultaneously process multiple different samples. The RNA extracted from this kit can be directly applied to RT-PCR Northern Blot、Dot Blot、 Experiments such as in vitro translation. Product Features
• High purity: maximally removes impurities such as proteins, and the extracted RNA can be directly used for various downstream experiments.
• Large extraction capacity: With a unique lysis buffer formula, cells or tissues can be fully lysed, and RNA extraction can reach up to 100 μ g.
Quick: With fewer steps, simple operation, and time-saving.
Strong compatibility: suitable for extracting RNA from various animal and plant tissues and cells. Self provided reagents:
70% ethanol (prepared without RNase water), anhydrous ethanol. Preparation and important precautions before the experiment. To prevent RNase contamination, attention should be paid to the following aspects:
1) Use RNase free plastic products and gun tips to avoid cross contamination.
2) Glassware should be dried and baked at a high temperature of 180 ℃ for 4 hours before use, while plastic utensils can be soaked in 0.5 M NaOH for 10 hours
Rinse thoroughly with water in minutes and then sterilize under high pressure.
3) The solution should be prepared using water without RNase.
4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.
2. The sample should avoid repeated freezing and thawing, otherwise it will affect the yield and quality of RNA extraction.
If TRIzon Reagent precipitates before use, it can be dissolved in a 56 ℃ water bath for a few minutes.
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 performed quickly.
6.If downstream experiments are highly sensitive to DNA, it is recommended to use DNase I without RNase to treat RNA. Usage method:
1. Sample processing
1a. Plant tissue: Take fresh plant tissue and grind it thoroughly in liquid nitrogen or cut it into pieces and quickly grind it directly in TRIzon Reagent. Add 1 mL of TRIzon Reagent to every 30-50 mg of tissue and mix well.
Note: The sample volume should not exceed 10% of the TRIzon Reagent volume.
1b. Animal tissue: Fresh or -70 ℃ frozen animal tissue should be cut into pieces as much as possible, and 1 mL of TRIzon Reagent should be added to every 30-50mg of tissue for homogenization treatment using a homogenizer. Or grind in liquid nitrogen and add 1 mL of TRIzon Reagent to mix well.
Note: The sample volume should generally not exceed 10% of the TRIzon Reagent volume.
1c. Single layer cultured cells: Remove the culture medium and directly add an appropriate amount of TRIzon reagent (1 mL TRIzon reagent is required per 10 cm ² area) to the culture plate. Use a sampler to repeatedly blow and beat the cells to lyse. Alternatively, after treatment with trypsin, transfer the cell solution to a RNase Free centrifuge tube, centrifuge at 300 × g for 5 minutes, collect the cell pellet, carefully aspirate all supernatant, add 1 mL of TRIzon Reagent and mix well.
be careful:
1) The number of collected cells should not exceed 1 × 107.
2) The dosage of TRIzon Reagent is determined by the area of the culture plate, not by the number of cells. If the dosage of TRIzon Reagent is insufficient, it may lead to DNA contamination in the extracted RNA.
3) When collecting cells, it is necessary to remove the cell culture medium completely, otherwise incomplete lysis may occur, resulting in a decrease in RNA production.
3) When collecting cells, it is necessary to remove the cell culture medium completely, otherwise incomplete lysis may occur, resulting in a decrease in RNA production.
1d. Cell suspension: Collect cells by centrifugation. Add 1 mL TRIzon Reagent every 5 × 10 ⁶ -1 × 10 ⁷ of animal, plant, and yeast cells, or every 10 ⁷ of bacterial cells.
be careful:
1) Do not wash cells before adding TRIzon Reagent to prevent RNA degradation.
2) Some yeast and bacterial cells may require homogenization or liquid nitrogen grinding treatment.
1e. Blood processing: Take fresh blood directly and add 3 times the volume of TRIzon reagent (recommended to add 0.75 mL of TRIzon reagent to 0.25 mL of whole blood), shake thoroughly and mix well.
1f. Optional steps: For samples with high protein, fat, polysaccharide, or extracellular substance content, such as muscle tissue, adipose tissue, or plant tubers, centrifugation at 12000 rpm (~13400 × g) for 10 minutes after homogenization treatment can be performed to remove insoluble substances. At this time, the precipitate contains extracellular substances, polysaccharides, and high molecular weight DNA, while RNA is present in the supernatant. 2. After adding TRIzon Reagent to the sample, blow it repeatedly several times to fully lyse the sample. Leave at room temperature for 5 minutes to completely separate the protein nucleic acid complex.
3. Add 200 μ L TRIzon PaI to every 1 mL TRIzon Reagent ™， Cover the tube tightly, shake vigorously for 15 seconds, and let it stand at room temperature for 2 minutes.
Centrifuge at 4.4 ℃ and 12000 rpm (~13400 × g) for 10 minutes. At this time, the sample is divided into three layers: a red organic phase, a middle layer, and an upper colorless aqueous phase. RNA is mainly in the upper aqueous phase. Transfer the upper aqueous phase to a new RNase Free centrifuge tube (provided).
5. Add an equal volume of 70% ethanol (prepared without RNase water) to the obtained aqueous solution, invert and mix well.
6. Add all the solutions obtained in the previous step into the Spin Columns RM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred in multiple times. Centrifuge at 12000 rpm for 20 seconds, discard the waste liquid from the collection tube, and place the adsorption column back into the recovery manifold.
7. Add 700 μ L Buffer RW1 to the adsorption column, centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the recovery header.
8. Add 500 μ L of Buffer RW2 to the adsorption column (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the recovery header.
9. Repeat step 8.
Centrifuge at 10.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes and let it dry thoroughly.
Attention: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as digestion, PCR, etc.).
11. Place the adsorption column in a new RNase free centrifuge tube, add 30-50 μ L RNase Free Water to the middle of the adsorption column, let it stand at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -70 ℃ to prevent degradation.
be careful:
1) The volume of RNase Free Wat should not be less than 30 μ L, as a small volume can affect the recovery rate.
2) If you want to increase RNA production, repeat step 11 with 30-50 μ L of 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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