Sample4 rxns ($16.8)
Sample4 rxns ($16.8)
Note: PureDireX / Purification Kits / Extraction Kit / Plant / RNA
The Plant Total RNA Isolation Reagent provides an easy 3-step method to isolate the total RNA from plant samples. This unique reagent system ensures the total RNA with a high yield and good quality from the most common plant samples as well as samples high in polysaccharides. If a larger sample is required, the kit volume can be scaled proportionately, making the kit not only very user-friendly but also highly versatile. The RNA phenol extraction is not required, and the entire procedure can be completed in 2 hours. The total RNA (up to 80 μg for fresh plant tissue) is ready for use in RT-PCR, Northern Blotting, cDNA Synthesis and Mapping.
|PR Buffer 1||100 ml|
|PR Buffer 2||10 ml|
1. Cut off 100 mg of the fresh plant tissue or 50 mg of the dry plant tissue.
2. Grind the sample in the liquid nitrogen to a fine powder using a mortar and pestle.
1. Add 1 ml of the PR buffer 1 and 12 µl of the ß-mercaptoethanol to the sample in the mortar and grind the sample until it is completely dissolved.
2. Transfer the dissolved sample to a 1.5 ml microcentrifuge tube.
3. Incubate at 70°C for 50 minutes.
4. Incubate at 15-30°C for 5 minutes.
5. Centrifuge at 2-8°C at 14-16,000 x g for 15 minutes.
6. Transfer the supernatant to a new 1.5 ml microcentrifuge tube.
1. Add a 1/10 volume of the PR buffer 2 and 500 μl of the chloroform to the supernatant from the Step 1.
2. Shake vigorously and then centrifuge at 2-8°C at 14-16,000 x g for 10 minutes.
3. Carefully remove the upper phase and transfer it to a new 1.5 ml microcentrifuge tube.
4. Repeat the Phase Separation Step until the interphase becomes clear, and then transfer the clear upper phase to a new 1.5 ml microcentrifuge tube.
NOTE: The number of repetitions is dependent on the sample type, e.g. dense tissue samples may require a higher number of repeats.
1. Add 500 μl of the isopropanol to the 1.5 ml microcentrifuge tube containing the clear upper phase from the Step 2.
2. Mix the sample by inverting gently and incubating on the ice for 10 minutes.
3. Centrifuge at 2-8°C at 14-16,000 x g for 15 minutes.
4. Discard the supernatant and wash the pellet with 1 ml of the 70% EtOH.
5. Centrifuge at 2-8°C at 14-16,000 x g for 5 minutes.
6. Completely discard the supernatant and re-suspend the pellets in 50-100 ul of the RNase-free H2O.
7. Incubate for 10 minutes at 60°C to dissolve the pellet.
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