| Product Description | This reagent kit is based on TRIzon's improved columnar total RNA extraction kit. This product can be extracted from animal groups
Extract total RNA from samples such as textiles, plant materials, various microorganisms, and cultured cells. Firstly, the cracking solution is fully cracked and
Homogenized samples, in their unique high salt state, RNA specifically binds to silicon matrix membranes, greatly reducing
Effectively removing organic solvent contamination while removing protein contamination, resulting in higher purity and quality of RNA. book
The product can quickly extract total RNA from various cells or tissues, and can process 30-50 mg of tissue or 5 × 10 ⁶ cells each time,
Can handle multiple different samples simultaneously. If it is an RNA experiment that is very sensitive to trace amounts of DNA, the residual DNA can be utilized
Using DNase without RNase for digestion and removal on the column, the extracted RNA can be directly applied to RT-PCR Experiments such as Northern Blot, Dot Blot, and in vitro translation.
| U665516 |
Component |
50 T |
Storage |
| U665516A |
DNase I |
1000 U |
-20℃. Avoid freeze/thaw cycle. |
| U665516B |
10×Reaction Buffer |
1000 μL |
-20℃. Avoid freeze/thaw cycle. |
| U665516C |
TRIzon Reagent |
60 mL |
2-8℃. Protect from light. |
| U665516D |
TRIzon PaI™ |
10 mL |
2-8℃. Protect from light. |
| U665516E |
Buffer RW1 |
40 mL |
RT |
| U665516F |
Buffer RW2 (concentrate) |
11 mL |
RT |
| U665516G |
RNase-Free Water |
10 mL |
RT |
| U665516H |
Spin Columns RM with Collection Tubes |
50 sets |
RT |
| U665516I |
RNase-Free Centrifuge Tubes (1.5 mL) |
50 EA |
RT | |
Preparation and important precautions before the experiment:
1.To prevent RNase pollution, attention should be paid to the following aspects:
1) RNase's plastic products and gun heads to avoid cross contamination.
2) Prepare the solution using water without RNase.
3) 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.
3. If TRIzon Reagent is found to have precipitates before use, it can be dissolved in a water bath at 56 ℃ 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 carried out quickly. Usage：
1. Sample processing
1a. Organization: 30-50 mg of tissue is thoroughly ground in liquid nitrogen and 1 mL of TRIzon Reagent is added, or 1 mL of TRIzon Reagent is added to the tissue sample and homogenized.
Attention:
The sample volume should not exceed 10% of the volume of TRIzon Reagent.
2a. Single layer cell culture: Remove the culture medium and add an appropriate amount every 10 cm ² Add 1 mL of TRIzon Reagent.
3a. Cell suspension: Collect cells by centrifugation. Add 1 mL of TRIzon Reagent to every 5 × 10 μ m cell.
2. After adding TRIzon Reagent, repeatedly blow a few times to fully crack the sample. Leave at room temperature for 5 minutes to completely separate the protein nucleic acid complex.
3. Add 200 to every 1 mL of TRIzon Reagent μ LTRIzon PaI ™， Cover the tube tightly, vigorously shake for 15 seconds, and let it sit at room temperature for 2 minutes.
4. Centrifuge at 4 ℃ 12000 rpm (~13400 × g) for 10 minutes. At this time, the sample is divided into three layers: the red organic phase, the middle layer, and the upper colorless aqueous phase. RNA is mainly in the upper aqueous phase. Move 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 to 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 batches. Centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
7. Add 350 to the adsorption column μ L Buffer RW1, centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.
8. Preparation of DNase I mixture: Take 52 μ LRNase Free Water, add 8 to it μ L 10 x Reaction Buffer and 20 μ L DNase I (1 U/ μ L) Mix well and prepare to a final volume of 80 μ The reaction solution of L.
9. Directly add 80 µ L DNase I mixture to the adsorption column and incubate at 20-30 ℃ for 15 minutes. 10. Add 350 to the adsorption column μ L Buffer RW1, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the recovery manifold.
11. Add 500 to the adsorption column μ L Buffer RW2 (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 collection tube.
12. Repeat step 11.
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 and thoroughly air dry.
Attention:
The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (enzyme digestion,...)PCR, etc. 14. Place the adsorption column in a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column 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 14 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 14.
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