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).
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.
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.
1. Add 300 l RNA hydration solu tion (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.
back to top