Chlamydomonas RNA extraction with Tri Reagent_15894433

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title: "TheNessLab : Chlamydomonas RNA extraction with Tri-Reagent"

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TheNessLab : Chlamydomonas RNA extraction with Tri-Reagent

Created by Josianne Lachapelle on Dec 05, 2017

(With modifications, from Adriano Senatore, adapted from Chomezynski and Sacchi 1987)

1. Pellet cells in a 2mL microcentrifuge tube.
2. Flash freeze pellet in liquid nitrogen, and store in -80C until time of extraction.
3. Add three small metal beads to the pellet.  Add 750 uL Tri Reagent to the pellet. Leave on ice.
4. Finely grind tissue using homogeniser.
5. Centrifuge mixture at 12 000 x g at room temperature for 10 minutes.
6. Transfer the supernatant to a new 1.5 mL tube.
7. Add 150 uL of chloroform. Vortex for 30 seconds and then leave for 10 minutes at room temperature.
8. Centrifuge mixture at 12 000 x g at room temperature for 10 minutes.
9. Repeat steps 4 to 6.
10. Transfer the supernatant to a new tube.
11. Add 375 uL of 2-propanol.
12. Incubate mixture for 10 minutes at room temperature.
13. Centrifuge for 10 minutes at 13 000 x g at 4°C.
14. Decant the supernatant and wash the pellet with 750 uL 70% (v/v) ethanol in RNA free distilled deionized water. Mix by inversion for 2 minutes.
15. Centrifuge at 13 000 x g at 4°C for 5 minutes.
16. Decant the ethanol let the pellet air dry.
17. Dissolve the pellet in 200 uL RNA free water.
18. Centrifuge at 13 000 x g at 4°C for 5 minutes.
19. Precipitate RNA by adding 43 mL of 10M lithium chloride for every 100 uL of sample. Leave at 4°C overnight.
20. The next day, centrifuge at 13 000 x g at 4°C for 20 minutes.
21. Decant the supernatant. Wash pellet with 500 mL of 70% ethanol (v/v) in RNA free water. Vortex.
22. Centrifuge at 13 000 x g at 4°C for 10 minutes.
23. Decant the ethanol and resuspend the pellet in 25-100 uL RNA free water.
24. Store at -80°C.

 

Document generated by Confluence on May 22, 2024 11:44

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