dsRNA inducing and extraction
1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 5 mL LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hours at 37°C with vigorous shaking (~ 300 rpm).
2. Adding the product into 50mL LB medium. Incubate for ~3-4 hours at 37°C with vigorous shaking (~ 300 rpm).
3. When the bacterial density between 2. 0 and 3. 0 at OD600, add 50μL IPTG（0. 5mol/L）Incubate for ~3-4 hours at 37°C with vigorous shaking (~ 300 rpm).
4. Centrifuge at 10,000 x g for 1 minutes at room temperature.
5. Decant or aspirate and discard the culture media.
6. Add 5mL Solution A. Vortex or pipet up and down to mix thoroughly. 5 minutes incubation is necessary.
7. Add 5mL Solution B. Vortex or pipet up and down to mix thoroughly.
8. Centrifuge at 10,000 x g for 15 minutes at room temperature.
9. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into a new centrifugal tube.
10. Add 5mL Solution C. Centrifuge at 10,000 x g for 15 minutes at room temperature.
11. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into a new centrifugal tube.
12. Add the same volume of isopropyl alcohol and 0. 5mL sodium acetate solution(3mol/L).
13. Ice-bath for 15 minutes.
14. Centrifuge at 10,000 x g for 15 minutes at room temperature.
15. Discard the supernatant.
16. Add 1 mL alcohol solution (70%). Vortex or pipet up and down to solute thoroughly. Transfer the solution to 1. 5mL Eppendorf tube.
17. Centrifuge at 13,000 x g for 10 minutes at room temperature.
18. Discard the supernatant and reuse the Eppendorf tube. Let sit at room temperature for about 20 minutes.
19. After the Eppendorf tube is totally dry, add 90μL RDD buffer, 10μLSSC buffer and 10μL DNase.
20. Let sit at room temperature for about 15 minutes.
21. Use agarose gel electrophoresis and gel extraction to pure dsRNA.
22. Store DNA at -20°C.
