Product Description
This kit can quickly extract high-quality total RNA from cells, bacteria, and some animal tissues without using toxic substances such as phenol and chloroform. The Lysis Buffer in the reagent kit contains strong denaturing agents and RNAse inhibitors, which can rapidly lyse the sample and inactivate RNAse, ensuring the integrity of RNA during the operation. The extracted total RNA can be used for various experiments such as RT-PCR, qPCR, Northern blotting, and cDNA library construction. The entire extraction process only takes 10 minutes, with simple and fast operation and high stability. This kit is only suitable for certain tissue types, including liver, intestine, brain, spleen, and soft tumor tissues. It is not suitable for tough tissues such as lungs, heart, skin, bones, muscles, etc.
ordering information
Product Name | Item number | Specifications |
Total RNA Column Extraction Kit | AN51L518 | 100T |
Product components
Components | 100T |
Lysis Buffer | 60 mL |
Wash Buffer* | 12 mL |
Elution Buffer | 10 mL |
RNA purification column (with collection tube) | 100 sets |
◆Wash BufferPlease add 48ml anhydrous ethanol before use，Shake well before use。
Transportation and storage
Transport at room temperature. Stored at room temperature, with a shelf life of 12 months.
usage method
1、 Sample cracking
1. Cells cultured on the wall
(1) Remove the culture medium and wash once with PBS;
(2) Add 500 μ L of Lysis Buffer (<3 × 10 ^ 6) to the culture plate
Place the cell horizontally for a moment, then blow or stir it several times with a gun until
Complete cell lysis. Transfer to a 1.5mL centrifuge tube, blow vigorously and repeatedly several times, fully lyse until no cell clusters are visible, and then proceed to
Step 2:;
Note: (1) Cell samples are recommended to be cultured in 6-well plates or 35mm culture dishes, and cultured to the appropriate density for RNA extraction (fine samples cultured in 24 well plates)
Cell density of over 90% can also be used for cell culture. (2) For culture containers that are not convenient for direct lysis, cells can be scraped off or
After trypsin digestion, collect the cells into centrifuge tubes. (3) For cells with small volume and low RNA content such as T cells/B cells, establish
Suggest increasing the number of cells to at least 1 × 10 ^ 6
above.
2. Suspension cultured cells
(1) Take 1~3 × 106
Transfer a suspension of cells to a 1.5 mL centrifuge tube and centrifuge at 1000 g for 1 minute to collect the cells;
(2) Remove the supernatant, add 500 μ L of Lysis Buffer, vigorously blow and suck 10 times, vortex for 10 seconds, to ensure complete cell lysis without visible cell clusters,
Then proceed to step two.
3. Bacterial cells
Centrifuge at 5000 rpm for 3 minutes to collect an appropriate amount of bacterial cells (<5 × 10 ^ 8)
）Discard the supernatant and add 100 μ L of TE buffer containing lysozyme. Blow and mix
Evenly incubate at room temperature for several minutes. Add 500 μ L of Lysis Buffer, vigorously shake for 20 seconds, centrifuge at 12000rpm for 1-2 minutes, take the supernatant, and then
Then proceed to step two。
4. Animal tissue (suitable for intestines, liver, brain, spleen, tumors, not suitable for hard tissues such as lungs, heart, skin, etc.)
(1) Homogenizer: Cut fresh tissue into 10-50mg to 1.5 mL centrifuge tubes, add 500 μ L Lysis Buffer, and thoroughly homogenize with a tissue homogenizer
Homogenize the tissue until there are no visible tissue blocks. Centrifuge at 12000 rpm for 1-2 minutes.
(2) Liquid nitrogen grinding: Grind 10-50mg of tissue thoroughly in liquid nitrogen, transfer the ground sample to a centrifuge tube, and add 500 μ L of
Lysis Buffer， Shake vigorously for 20 seconds and centrifuge at 12000rpm for 1-2 minutes.
(3) Transfer no more than 400 μ L of supernatant into a new centrifuge tube. Do not suck sediment and foam at the bottom of the pipe.
2、 RNA extraction
1. Cell samples
Accurately estimate the volume of the lysate, add an equal volume of anhydrous ethanol to the cell lysate, vigorously invert the centrifuge tube several times, or use a pipette
Blow and suck vigorously several times, mix thoroughly, then transfer the liquid to a centrifuge column and proceed to step 3. [Note]: Precipitation may occur, which is a normal phenomenon
, does not affect the extraction process, continue with subsequent operations.
2. Organize samples
Accurately estimate the volume of the lysate, add 0.5 times the volume of anhydrous ethanol (or an equal volume of 70% ethanol) to the lysate, and vigorously centrifuge the tube
Invert several times, or use a pipette to forcefully blow and suck several times, mix thoroughly, and then transfer the liquid onto a centrifuge column.
3. Centrifuge at 7000 rpm for 1 minute (if there is still liquid residue on the centrifuge column, increase the speed to 12000 rpm), and discard the waste liquid.
4. Add 500 μ L of Wash Buffer to the RNA column and centrifuge at 12000 rpm for 1 minute.
5. Discard the waste liquid, install the RNA column into the recovery manifold, centrifuge the empty tube at 12000 rpm for 1 minute, and completely remove any residual Wash Buffer.
6. Remove the RNA adsorption column and place it in a 1.5mL RNase free centrifuge tube. Open the lid and air dry for 1 minute. Add to the membrane center of the RNA column
25-50uL Elution buffer, let it stand at room temperature for 1 minute.
Centrifuge at 12000 rpm for 1 minute. The sample can be stored for a long time until -80 ℃. [Note]: The volume of eluent added should not be less than 25uL, otherwise it will affect the recovery
Yield: In order to obtain a higher concentration of RNA, the centrifuged RNA solution can be re added to the adsorption column and eluted repeatedly.
matters needing attention
1. This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields.
2. After adding Lysis Buffer, it is necessary to shake it thoroughly to ensure the effectiveness of RNA extraction; If the mixture is very viscous and difficult to transfer, it is obvious that the sample
Excessive usage, increase Lysis buffer usage or reduce sample usage
3. Cell or tissue lysate products need to be added with anhydrous ethanol before being loaded onto the column, thoroughly mixed, and then centrifuged on a centrifuge column.
4. The samples used should avoid repeated freezing and thawing to avoid affecting the yield and quality of RNA.
5. Regarding RNA purity: OD260/OD280 and OD260/OD230 ratio are indicators for measuring RNA purity. High quality RNA,
The value of OD260/OD280 is between 1.9-2.2, and OD260/OD230 is greater than 1.8 (a relatively pure ratio greater than 2.0). This reagent kit extracts
RNA， Using a micro spectrophotometer such as Nanodrop to measure OD 260/280 between 1.90-2.2 and OD 260/230 between 2.0-2.2, both belong to the same category
Normal.
Related product recommendations
Total RNA Extraction Kit (Trizol) (Product Code: AN51L758)
RNA Extraction Auxiliary Reagent (Chloroform Substitute) (Product Code: AN51L677)
This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields.
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