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Protocol for RNA Isolation Using TRIzol Reagent with Phase Lock ,,, Gel-Heavy

to collect gel at tube bottoms).
  • Add cell lysate to the tubes containing pre-spun Phase Lock Gel-Heavy and incubate 5 minutes at 1530C.
  • Add 0.2 ml chloroform (or chloroform-isoamyl alcohol) per 1 ml TRIzol Reagent initially used. Cap tubes and shake vigorously for 15 seconds. DO NOT VORTEX!
  • Centrifuge samples at no more than 12,000 x g for 10 minutes at 28C.
    NOTE: 15 ml and 50 ml PLG-H screw-cap tubes should be centrifuged at or below 2000 x g.
  • Examine phasing. Clear, aqueous phase should be entirely atop Phase Lock Gel. The phenol-chloroform phase and cloudy interphase should be below Phase Lock Gel layer. If this is not the case, add another 0.2 ml chloroform (or chloroform-isoamyl alcohol) per 1 ml TRIzol Reagent used initally and shake vigorously. Repeat centrifugation and re-examine phasing.
  • Transfer aqueous phase containing RNA to a fresh tube (aqueous phase may be decanted).
  • Precipitate RNA by adding 0.5 ml Isopropyl alcohol per 1 ml TRIzol Reagent used initially. Mix samples by repeated inversion. Allow samples to incubate at 1530C for 10 minutes. Centrifuge samples for 10 minutes at no more than 12,000 x g, 28C. RNA pellet should be visible on side and bottom of tube.
  • Decant supernatant. Add 1 ml 75% ethanol per 1 ml TRIzol Reagent used initially to wash the RNA pellet. Mix samples to dislodge pellet, using a vortex if necessary. Centrifuge samples at no more than 7,500 x g for 5 minutes at 28C.
  • Carefully decant supernatant. Briefly air-dry or vacuum-dry the RNA pellet to remove residual ethanol (5
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