Orcinol Determination of RNA
OrcinolDeterminationofRNALEVELI
Materials
- RNA
- Alkalinedistilledwater
- Acid-orcinolreagent
- Boilingwaterbath
- Spectrophotometerandcuvettes
Procedure
- PrepareaseriesofseriallydilutedRNAstandardsasinExercise14.8,buthavingarangefrom1.0mg/mldownto0.125mg/ml.
- PrepareaserialdilutionofyoursampleRNAasinExercise14.8.
- Place3.0mlofeachstandardand3.0mlofeachserialdilutionofthesampleRNAintoseparatetesttubes.Place3.0mlofalkalinewaterinaseparatetube.
- Add3.0mlofacid-orcinolreagenttoeachtubeandmixwell.
- Add0.3mlofalcohol-orcinolreagenttoeachtubeandmixwell.
- Placethetubesinaboilingwaterbathfor20minuteswithmarblesplacedontopofeachtubetopreventevaporation.Coolthetubesbyimmersioninanicebathattheendofthe20minuteperiod.
- Turnonaspectrophotometerandadjustthewavelengthto660nm.Blankthespectrophotometerwiththealkalinewater/orcinoltube.MeasuretheA
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ofeachoftheremainingstandardsanddilutedsamples. - Plottheabsorbanceofthestandardsagainsttheknownconcentrations.Calculatetheextinctioncoefficient,andcalculatetheconcentrationofRNAinyoursample.Usethedilutionyieldinganabsorbancebetween0.1and1.5absorbanceunits.
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