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

Phase Lock Gel may be used in conjunction with TRIzol Reagent for the isolation of total RNA from cell and tissue samples. Increased yields are observed using this method, because the entire aqueous phase can be recovered without interphase contamination. Below is a protocol outlining the steps involved in RNA isolation with TRIzol Reagent and Phase Lock Gel-Heavy. For further details about TRIzol Reagent, please refer to TRIzol Reagent protocol.

IMPORTANT: When working with TRIzol Reagent, use appropriate protective clothing and work under a chemical fume hood.

  1. Homogenization:
    1. Tissues: Add 1 ml TRIzol Reagent per 50100 mg tissue and homogenize with a POLYTRON homogenizer. For 110 mg quantities of tissue, add 0.8 ml TRIzol Reagent and 0.2 ml 1 mg/ml mussel glycogen and homogenize.
    2. Cells grown in a Monolayer: Lyse cells by adding 1 ml TRIzol Reagent per 10 cm2 area of culture dish. Pipette suspension several times to disrupt cells. For 102104 cells, add 0.8 ml TRIzol Reagent and 0.2 ml 1 mg/ml mussel glycogen and homogenize.
    3. Cells grown in Suspension: Pellet cells by centrifugation. Do not wash cell pellet. Add 1 ml TRIzol Reagent per 510 x 106 animal, plant, or yeast cells or 1 x 107 bacterial cells. Resuspend pellet by pipetting to lyse cells. For 102104 cells, add 0.8 ml TRIzol Reagent and 0.2 ml 1 mg/ml mussel glycogen and homogenize.
  2. Pre-spin the appropriate sized Phase Lock Gel-Heavy tubes briefly to collect gel on tube bottoms (1500 x g for 30 seconds is sufficient
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