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Total RNA Isolation From 50100 mg Plant Tissue


AquaPure RNA Isolation Kit
Catalog #732-6370
Expected yield range: 50200 g RNA


Method
Cell Lysis
1.Add 50100 mg (0.050.1 g) frozen ground tissue or fresh tissue to a tissue grinder tube on ice and then add 3 ml cell lysis solution. Grind frozen tissue finely in liquid nitrogen with a porcelain mortar and pestle. Keep tissue frozen until added to the tissue grinder tube.

2 . Homogenize quickly using 510 strokes with a tube pestle. Transfer sample to an Oak Ridge centrifuge tube (or other tube rated for high speed).

Protein-DNA Precipitation
1 . Add 1 ml protein-DNA precipitation solution to the cell lysate.

2.Invert tube gently 10 times and place tube into an ice bath for 10 min.

3.Centrifuge at 15,000 x g for 5 min. The precipitated proteins and DNA will form a tight pellet.

RNA Precipitation
1. Pour the supernatant containing the RNA (leaving behind the precipitated protein-DNA pellet) into a clean Oak Ridge centrifuge tube (or other tube rated for high speed) containing 3 ml 100% isopropanol (2-propanol).

2. Cap the tube. Mix the sample by inverting gently 50 times.

3. Centrifuge at 15,000 x g for 5 min; the RNA will be visible as a small, translucent pellet.

4. Pour off supernatant and drain tube on clean absorbent paper. Add 3 ml 70% ethanol. Cap and invert the tube several times to wash the RNA pellet.

5. Centrifuge at 15,000 x g for 2 min. Carefully pour off the ethanol.

6. Invert and drain the tube on clean absorbent paper and allow to air-dry for 15 min.

RNA Hydration
1. Add 300 l RNA hydration solution (300 l will give a concentration of 500 g/ml if the total yield is 150 g RNA).

2. Allow RNA to rehydrate on ice at least 30 min. Vortexvigorously for 5 sec, pulse-spin and carefully transfer sample to a 1.5 ml microfuge tube. Store purified RNA sample at - 7 0 C t o -80C until use.

3. Before use, vortex sample vigorously for 5 sec and pulsespin. Pipet sample up and down several times to ensure adequate mixing.



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