Trizol extracting RNA from cells

Materials

  • 1X SDS extraction buffer (Rio)
    • 0.5% SDS
    • 1mM EDTA
    • 20mM Tris pH 7.5

    1. Add trizol directly to dish and homogenize by trituration
    DishTrizol volume (ul)
    12 well100
    6 well300
    10cm1000
    1. Incubate 5 minutes at RT
    1. Add 1 volume chloroform per 5 volumes trizol and shake gently (e.g. 0.2mL chloroform per 1mL trizol)
    DishChloroform volume (ul)
    12 well20
    6 well66
    10cm200
    1. Rest two minutes at RT
    1. Spin 10k g for 10 minutes at 4 degrees
    1. Move the aqueous (clear) phase to a new tube (volume of this should be ~50% total volume from step 4)
    1. Add 1 volume isopropanol per 2 volumes of the aqueous phase (e.g. 0.5mL isopropanol to 1mL aqueous phase)
    1. Mix by shaking
    1. Rest 10 minutes at RT
    1. Pellet RNa at 10k g for 10 minutes at 4 degrees
    1. Discard supernatant
    1. Resuspend RNA in 50µL SDS extraction buffer
    1. Add 250µL 0.3M NaOAc
    1. Add 4µL glycoblue
    1. Add 750µL 100% EtOH
    1. Put in -80 for overnight.
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