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 4oC.

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 4oC.

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 4oC.

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 60oC to dissolve RNA.
 * 3) Store at -80oC.