Kai Yuet/Protocols:RNA Isolation

RNA Isolation (KPY)

Materials

• Chloroform
• 75% Ethanol
• Isopropyl Alcohol
• RNase-free Water
• TE Buffer
• TRIZOL Reagent

Procedures

Homogenization

1. Lyse cells by adding 500 µL of TRIZOL directly to the dish, mixing and passing the cell lysate several times by pipetting.

Phase Separation

1. Incubate the samples for 5 minutes in Eppendorfs at room temperature.
2. Add 100 µL of chloroform per 500 µL of TRIZOL.
3. Vigorously shake the microcentrifuge tubes by hand for 15 seconds.
4. Incubate the tubes for 2 to 3 minutes at room temperature.
5. Centrifuge the tubes at 12000 x g for 15 minutes at 4^oC.

RNA Precipitation

1. Transfer the aqueous phase (60% TRIZOL by volume) to a fresh Eppendorf.
2. Add 250 µL of isopropyl alcohol per 500 µL of TRIZOL to precipitate RNA.
3. Incubate the tubes for 10 minutes at room temperature.
4. Centrifuge the tubes at 12000 x g for 10 minutes at 4^oC.

RNA Wash

1. Remove the supernatant.
2. Resuspend the pellet in 500 µL 75% ethanol per 500 µL TRIZOL and vortex.
3. Centrifuge the tubes at 7500 x g for 5 minutes at 4^oC.

Redissolving the RNA

1. Air dry the RNA pellet and dissolve with TE buffer (200 µL).
2. Incubate for up to 10 minutes at up to 60^oC to dissolve RNA.
3. Store at -80^oC.

Notes

• Partially dissolved RNA have an A_260/280 < 1.6.

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