Team:Wageningen UR/Protocol/RNA

From 2012.igem.org

Raymond’s Plant RNA Extraction Protocol

PROTOCOL

1. Grind approx 0.1 g tissue in liquid nitrogen.

2. Add 1 mL of Trizol reagent to the ground powder (see Note 5).

3. Transfer into Eppendorf tubes.

4. Centrifuge at 12,000g for 5 min at 2 to 8°C.

5. Remove supernatant to new Eppendorf tube.

6. Add 200 μL of chloroform and shake vigorously by hand for approx 15 s.

7. Let stand at room temperature (~20–25°C) for 3 min.

8. Centrifuge at 12,000g for 15 min at 2 to 8°C.

9. Transfer the upper aqueous phase to a new tube (ensure no interface debris is transferred!!). (If you want to see the upcoming RNA pellet better, add ~10ug Glycogen!)

10. Add 0.5 mL of isopropyl (=isopropanol = 2-propanol) alcohol. Mix.

11. Let stand at room temperature for 10 min.

12. Centrifuge at 12,000g for 10 min, at 2 to 8°C.

13. Carefully discard supernatant (tip Eppendorf with the pellet position angled up and away from you and pipet out the supernatant). The pellet may be slightly glassy and transparent or may not be clearly visible at all.

14. Add 1 mL of 75% ethanol.

15. Vortex briefly and centrifuge at 12,000g for 5 min at 2 to 8°C.

16. Discard the supernatant as in step 13 and allow pellet to air-dry for 10 min.

17. Dissolve the pellet in 20 μL of RNase-free water (=MQ straight from machine) by very gently sucking the liquid up and down with a pipet.

18. Measure the yield on the Nanodrop (do not forget the proper settings, note the A260/A280)

19. Store at -20

Check RNA quality on a denaturing Ethidiumbromide gel.

The 28s rRNA band appears equal to or more abundant than the 18s rRNA band, thereby indicating that little or no RNA degradation occurred during extraction.