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HOME > Protocols > Molecular Biology > RNA > Protocol for RNA Isolation from Tissue Samples

Protocol for RNA Isolation From Tissue Samples
(Chomczynski and Sacci, BioAnalytical Chemistry 162: 156-159, 1987)

    1. Homogenize tissue with Solution D (1ml solution D/100mg tissue).
    2. Add 2M Sodium Acetate (0.1ml/100mg tissue).
    3. Mix by inversion.
    4. Add Phenol equilibrated to ~pH 4.5 (1ml/100mg tissue).
    5. Mix by inversion.
    6. Add Chloroform (0.2ml/100mg tissue).
    7. Mix by inversion.
    8. Vortex vigorously for 10 seconds.
    9. Cool on ice.
    10. Centrifuge at 10,000xg for 20 minutes at 4oC.
    11. Transfer aqueous phase to a fresh tube.
    12. Add isopropanol (1ml isopropanol/100mg tissue).
    13. Vortex briefly.
    14. Incubate at -20oC for at least 1 hour.
    15. Centrifuge at 10,000xg for 20 minutes at 4oC.
    16. Discard supernatant.
    17. Dissolve pellet in Solution D (0.3ml/100mg tissue).
    18. Add Isopropanol to precipitate (0.3ml/100mg tissue).
    19. Incubate at -20oC for at least 1 hour.
    20. Centrifuge at 10,000xg for 20 minutes at 4oC.
    21. Discard supernatant.
    22. Wash pellet with 70% ethanol.
    23. Centrifuge at 10,000xg for 10 minutes at 4oC.
    24. Air-dry the pellet for 10 minutes.
    25. Dissolve pellet in desired buffer (50l/100mg tissue).

Denaturing solution - Solution D (add 360l beta-mercaptonethanol to 50ml immediately before use): 4 M Guanidine (isothiocyanate), 25 mM Sodium Citrate, 0.5% SDS.


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