Sample4 rxns ($31.2)
Sample4 rxns ($31.2)
Note: PureDireX / Purification Kits / Extraction Kit / RNA / Tissue
The Total RNA Isolation Kit (Tissue) provides a fast, simple, and cost-effective method for isolation of total RNA from the tissue sample. Detergents and chaotropic salt are used to lyse cells and inactivate RNase. The specialized high-salt buffering system allows RNA species bases to bind to the glass fiber matrix of the spin column while contaminants pass through the column. Impurities are efficiently washed away, and the pure RNA is eluted with REL Buffer without phenol extraction or alcohol precipitation. RNA purified with The Total RNA Isolation Kit is suitable for a variety of routine applications including RT-PCR, cDNA Synthesis, Northern Blotting, Differential display, Primer Extension and mRNA Selection. The entire procedure can be completed within 25-40 minutes.
|Buffer RR||45 ml||2 ml|
|Buffer W1||45 ml||2 ml|
|Buffer W2 (Add ethanol)||15 ml (60 ml)||300 µl × 2 (1.5 ml × 2)|
|Buffer REL||10 ml||1 ml|
|Column RT||100 pcs||4 pcs|
|Collection Tubes||100 pcs||4 pcs|
1. Cut off up to 30 mg of fresh or frozen animal tissue and grind the sample under liquid nitrogen to a fine powder using a mortar and pestle. (If using frozen animal tissue, the sample MUST have been flash frozen in liquid nitrogen and immediately stored at -70°C until use, to avoid RNA Degradation).
2. Proceed with the Step2 Lysis.
1. Slice small sections (up to 25 mg) from blocks of paraffin-embedded tissue and transfer to a 1.5 ml microcentrifuge tube.
2. Add 1 ml of xylene to the tube.
3. Vortex vigorously and incubate at room temperature for approximately 10 minutes.
4. Vortex occasionally during incubation.
5. Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
6. Add 1 ml of absolute ethanol to wash the sample pellet and mix by inverting.
7. Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
8. Add 1 ml of absolute ethanol to wash the sample pellet again and mix by inverting.
9. Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
10. Open the tube and Incubate at 37°C for 15 minutes to evaporate any ethanol residue.
11. Proceed with the Step 2 Lysis.
Refer to the table below to troubleshoot problems that you may encounter when purifying total RNA.
|Degraded RNA/ low integrity||RNases contaminant||Clean everything, use barrier tips, wear gloves and a lab coat, and use RNase-free enzymes, EX: RNase inhibitor.|
|Low yields of RNA||Incomplete lysis and homogenization||Use the appropriate method for the lysate preparation based on the amount of the starting materials immersed in the Buffer RA to achieve the optimal lysis.|
|Incorrect elution conditions||Add 50 μl of the RE Buffer to the center of each Column DR, let it stand for 2 minutes, and centrifuge at 14,000 x g for 2 minutes.|
|Inhibition of downstream enzymatic reactions||Presence of ethanol in the purified RNA||Repeat the wash step: Centrifuge at 14,000 x g again for 2 minutes to remove the residual W2 Buffer.|
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