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Total RNA Isolation Protocol 1,2,3

repeated gentle inversion. Do not vortex.
  • Incubate on ice for 10 minutes.
  • Centrifuge at 12,00016,000 x g for 5 minutes in a microcentrifuge to separate the phases.
  • Add 200 l (100 l per sample from 48 or 96 well plates) Phenol-Chloroform-Isoamyl Alcohol (PCI, 50:49:1) to the aqueous phase in the same PLG tube. Mix thoroughly by repeated gentle inversion. Do not vortex.
  • Centrifuge at 12,00016,000 x g for 5 minutes to separate the phases.
  • Collect resultant aqueous phase to an RNase-free microcentrifuge tube, add an equal volume of 100% Isopropanol, and mix by repeated inversion.
  • Centrifuge at 12,00016,000 x g for 20 minutes.
  • Discard resultant supernatant and wash pellet several times with 200 l 70% Ethanol, using 23 minutes at 12,00016,000 x g to re-pellet the sample.
    Note: Samples may be stored in the 70% Ethanol wash at this stage at 70C or colder for extended periods.
  • Discard final wash and dry pellet at room temperature.
  • Re-dissolve pellet in a suitable volume (510 l) of RNase-free water. Store the RNA solution at 70C.
    Note: Absorbance determinations should be performed in RNase-free TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0).15
    • References
    1. Chirgwin, J.M. et al. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem. 18:5294-5299.
    2. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guan
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