RNA Extraction--RNA提取
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All solutions used in RNA preparation should be treated with DEPC as follows: add DEPC as follows: add DEPC to 0.1% and vigorously stir for one hour to O/N followed by autoclaving for 30 minutes. Bake all glassware. Soak microfuge tubes and corex tubes in a beaker of DEPC water, or fill with DEPC water and place on nutator O/N, follwed by autoclaving.
Keep RNA prep on ice unless otherwise indicated.
1. Grind tissue wit baked mortor and pestle in liquid nitrogen. (5g of tissue will fill 2 ultracentrifuge tubes);
2. Add 5 volumes (v/w) homogenization buffer (4M guanidine thiocyanate, 25mM sodium citrate pH 7.0, 1.5% sarcosyl-0.1 M 2- mercaptoethanol added just prior to using) to ground tissue in a beaker or sterile Falcon tube and homogenize with polytron grinder at setting 6 for 2 minutes;
Note: guanidine thiocyante is NASTY-wear goggles, take care (wash/run polyton with EtOH and DEPC's water between sample)
3. Pour into corex tubes and spin in SS34 rotor at 12K rpm for 10 minutes.
4. Lay supernatant gently over 3.6ml CsCl cushion (5.7M CsCl, 0.1M EDTA, pH 7.0) in ultracentrifuge tube(s) and *BALANCundefined; Note: transfer only clear liquid
5. Ultrancentrifuge O/N at 32K rpm (SW41T1) at room temperature
6. Vacuum suck, pipette, or pour off the liquid and set upside down 1-2 minutes to drain off liquid- suggest cutting off top of tube above pellet to avoid contamination with plant crud
7. 70% EtOH was pellet 2-3 times
8. Resuspend the pellet in DEPC-treated dH2O or TE buffer (10mM Tris, 1mM EDTA, pH 8.0) and transfer to microfuge tube
9. EtOH precipitate the RNA by adding 0.1 colume of 3M sodium acetate (pH 5.2) and 2.5-3 volumes of EtOH. Spin 10-15 minutes, 4℃. Spin dry minimally in speed vac. Resuspend pellet in TE buffer or dH2O.
10. Determine the RNA concentration at 260nm:
RNA conc. (µg/ml)= OD at 260nm x dilution factor x 40
DNA conc. (µg/ml)= OD at 260nm x dilution factor x 50
RNA should be used prompltly or alliquoted and stored at -70℃, avoid freeze/thaws.
