Mix 293 ml of water, 17.6 ml of 0.75 M sodium citrate (pH 7.0), and 26.4 ml of 10% (w/v) N-lauroylsarcosine. Add 250 g of guanidine thiocyanate. Dissolve guanidine thiocyanate at 60 C. Add 0.07 ml of 2-mercaptoethanol to 10 ml of the stock solution.
1. Grind frozen plant tissue in liquid nitrogen with motor and pestle.
2. Add to a conical tube10 ml of denaturing solution per 1 g of tissue.
3. Transfer the fine powder of the plant tissue into a conical tube. Keep the tissue powder frozen until it is transfered into the denaturing solution. Mix well so that the powder is dispersed in the denaturing solution as soon as possible.
4. Add 1 ml of sodium acetate (pH 4.0). Vortex for 1 min.
5. Add 10 ml of water-saturated phenol. Do not use buffer-saturated phenol. Mix thoroughly.
6. Add 2 ml of chloroform. Mix throughly, and stand the tube on ice for 15 min.
7. Centrifuge for 20 min at 10,000 x g. Transfer the aquaous phase into a clean tube. Do not try to remove all of the aquaous phase.
8. Add 10 ml of isopropanol. Chill the sample for 30 min at -20 C. Centrifuge for 10 min at 4 C. Discard the supernatant.
9. Dissolve the RNA pellet in 0.3 ml of denaturing solution. Transfer the sample into a clean microfuge tube.
10. Add 0.3 ml of isopropanol. Chill for 30 min at -20 C. Centrifuge for 10 min at 4 C. Discard the supernatant.
11. Resuspend the RNA in 75% ethanol. Vortex. Centrifuge for 10 min at 4 C. Discard the supernatant.
12. Dissolve the RNA in 0.3 ml of RNase-free water.
13. Extract the sample with an equal volume of phenol-chloroform. Centrifuge for 5 min at 4 C. Transfer the aquaous phase into a new tube. If interphase is visible, repeat phenol-chloroform extranction until no interphase is visible
14. Extract the sample with an equal amount of chloroform. Centrifuge for 5 min at 4 C.
15. Add 30 ul of 3 M sodium acetate and 1 ml of ethanol. Chill for 30 min at -20 C. Centrifuge for 10 min at 4 C. Discard the supernatant.
16. Rinse the pellet with 1 ml of 75% ethanol. Centrifuge for 5 min at 4 C. Discard the supernatant.
17. Dissolve the RNA in RNase-free water.
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