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).