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The Laboratory of J. Michael Bishop at the University of California, San Francisco
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1. Dissolve 50 mg Yeast RNA in 25 to 50 ml of DEPC Treated ddH2O.
2. Add an equal volume of Phenol, mix by inversion, centrifuge to separate the phases and save the upper (aqueous) phase.
3. To the aqueous phase, repeat the Phenol extraction (as in Step #2).
4. To the aqueous phase add an equal volume of Phenol:Chloroform, mix by inversion, centrifuge to separate the phases and save the upper (aqueous) phase.
5. To the aqueous phase add an equal volume of Chloroform, mix by inversion, centrifuge to separate the phases and save the upper (aqueous) phase.
6. To the aqueous phase add 2 volumes 100% Ethanol and 0.1 volume of 3 M Sodium Acetate.
7. Mix by inversion, centrifuge to pellet the RNA and decant the supernatant.
8. Dry the RNA pellet by inverting the tube over paper towel and allow to stand at room temperature for 10 min.
9. Resuspend the RNA pellet in 5 ml DEPC Treated ddH2O.
10. Analyze RNA for purity (see protocol on Quantification of RNA).
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| DEPC Treated ddH2O |
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| 3 M Sodium Acetate |
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| DEPC Treated ddH2O |
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| Phenol:Chloroform |
| 24:1 Phenol:Chloroform
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| DEPC Treated ddH2O |
| Shake vigorously and incubate overnight at room temperature
0.1% (v/v) Diethyl Pyrocarbonate (DEPC)
in ddH2O
Autoclave to inactivate DEPC
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| DEPC Treated ddH2O |
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Sodium Acetate
Chloroform
Ethanol
Phenol
RNA, Yeast
Diethyl Pyrocarbonate (DEPC)
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No hints are associated with this bioProtocol
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