Kafatos:totalRNA extraction from adult mosquitoes
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totalRNA extraction from adult mosquitoes
- Add 9 volumes TRIZOL to 1 volume of tissue (400μl of Trizol for 1-20 mosquitoes works fine)
- Homogenize
- Spin for 10 min at 12 krpm, 4˚C and transfer clear supernatant into a fresh tube.
- Incubate at RT for 5 min
- Add 0.2 ml CHCl3 per 1 ml Trizol. Shake vigorously by hand and incubate at RT for 5 min.
- Spin 15 min at 4˚C, 12 krpm. Transfer aqueous (upper) phase into a fresh tube.
- Add 0.5 ml Iso-propanol per 1ml Trizol, mix and incubate for 10 min @ RT
- Spin for 10 min @ 4˚C, 12 krpm.
- Use these steps to clean up the sample for more sensitive reactions:
- a. remove supernatant and resuspend in 300μl ddH2O
- b. add 30μl 3M NaOAc pH 3
- c. precipitate with 600μl abs. EtOH 15 min @ -80˚C
- Use these steps to clean up the sample for more sensitive reactions:
- 9. Remove supernatant and wash pellet with 1ml 75% EtOH per 1ml Trizol. Vortex.
- 10. spin for 5 min @ 4˚C, 8 krpm
- 11. Air- or speed-vac-dry pellet and dissolve RNA ~70μl H2O
try these tips from Antonio to increase final yield: step 7: use 0.8 volumes Iso-propanol per 1 volume Trizol, mix and precipitate 30 min @ -80˚C step 8: spin 30 min @ @ 4˚C, max krpm.