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Easy preparation of total RNA from cultured cells with TRIzol and ,,, Eppendorf Phase Lock Gel

d reaction tubes.

  1. Aspirate and discard culture medium from culture vessel.
  2. Add 3 ml of TRIzol reagent (Invitrogen) for 2 x107 cells.
  3. Incubate homogenized samples for 5 minutes at 15C to 30C.
  4. Aliquot lysate into five 2 ml PLG Heavy tubes and five 2 ml standard tubes without PLG (300 ml each).
  5. Add 60 l of chloroform/isoamyl alcohol (in a ratio of 49:1, v/v), cover samples tightly and shake vigorously for 15 seconds. Incubate at room temperature for 2 to 3 minutes.
  6. Centrifuge samples for 15 minutes at 12,000 x g at 4C.
  7. Transfer upper aqueous phase to a fresh tube.
  8. Precipitate RNA from aqueous phase by mixing with 150 l isopropanol. Store samples at room temperature for 10 minutes and centrifuge at 12,000 x g for 10 minutes. Remove supernatant and wash RNA pellet with 300 l 75% ethanol. Next, centrifuge at 7,500 x g for 5 minutes at room temperature.
  9. Remove the ethanol wash and briefly air-dry RNA pellet. Dissolve RNA in 100 l Molecular Biology Grade Water (Eppendorf).
RT-PCR*
  1. Heat 3 g of total RNA with 100 ng of oligo-dT-primer for first strand synthesis to 75C for 10 minutes in a reaction volume of 18.5 l. Chill tube on ice for 5 minutes and spin.
  2. Add 2.5 l dNTPs (2 mM), 2.5 l 10X RT-buffer and 1.5 l Reverse Transcriptase (300 U) to sample and incubate for 1.5 hours at 42C.
  3. Use 2 l of first strand synthesis as template for the PCR amplification of an 844 bp fragment of the human p53 gene.
    Amplification was performed in a final reaction volume of 25 l. PCR was p
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