Kafatos:totalRNA extraction from cells

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totalRNA extraction from cells (in 6-well plate) with Trizol

1. Remove medium and wash 2x with 5ml PBS.

2. Remove all traces of PBS and lyse the cells by adding 0.5 ml Trizol. Pass several times through a pipet.

3. Incubate homogenates for 10 min at RT.

it is possible to interrupt here and store the tubes at -80˚C

4. Add 100μl CHCL3 (Chloroform) per 0.5ml Trizol. Mix by shaking vigorously for 15" (by hand).

5. Centrifuge samples at 12krpm, 4˚C for 15min.

6. Transfer the colorless upper phase into a fresh tube and add 300μl iso-propanol.

7. Mix well and incubate at RT for 15min.

8. Spin at max speed, 4˚C for 15min.

9. Remove supernatant and wash pellet once with 1ml 70% Ethanol.

10. Mix by vortexing and spin at max speed for 5min, 4˚C.

11. Remove supernatant and dry pellet (in speed-vac or 37˚C).

12. Resuspend pellet in 50μl H2O (RNAse free).

13. Run 1μl on >1.5% agarose gel and determine the OD at 260nm (dilute ~1:200 if not using the Nanodrop).