NucleotideCompositionofRNA

LEVELII

Materials

• RNAsample
• 1Nand0.1NHCl
• Boilingwaterbath
• Whatman#1filterpaper(forchromatography)
• Chromatographytank
• 20µlmicroPipette
• Aceticacid:butanol:water(15:60:25)solvent
• UVlightsource
• UVspectrophotometer

Procedure

1. PlaceaportionofyourRNAsample(approximately40mg,hydrated)intoaheavywalledpyrextesttube.Add1.0mlof1NHClandsealthetube.

2. Heatthetubeinaboilingwaterbathfor1hour.

3. Coolthetube,openitandplacethecontentsintoacentrifugetube.Centrifugethecontentsat2,000RPMinaclinicalcentrifugetoremoveanyinsolubleresidue.ThesupernatantcontainsyourhydrolyzedRNA.

4. PrepareWhatmanfilterpaperNo.1forstandardone-dimensionalchromatography.12

5. Usingamicropipette,spot20µlofyourhydrolyzateontothepaper,beingcarefultokeepthespotsassmallaspossIBLe(repeatedsmalldropsarebetterthanonelargedrop).Allowthespotstocompletelydrybeforeproceeding.

6. Placethepaperchromatogramintoyourchromatographytankandaddthesolvent(aceticacid:butanol:water).Allowthesystemtofunctionforanappropriatetime(approximately36hoursfora20cmdescendingstripofWhatman#1).Removethepaperanddryitinacirculatingairovenat40°Cforabout2hours.

7. Locatethespotsofnucleotidesbytheirfluorescenceunderanultra-violetlightsource.ExposethepaperchromatogramtoaUVlightsourceandoutlinethespotsusingalightpencil.Theorderofmigrationfromthepointoforiginisguanine(lightbluefluorescence),adenine,cytilicacidandfinally,uridylicacid.

   DonotlookdirectlyattheUVlightsource.UseacABInetdesignedtoshieldfromharmfulUVrADIation.

8. Aftercafefullymarkingthespots,cutthemoutwithscissorsandplacethepapercutoutsintoseparatelylabeled15mlconicalcentrifugetubes.Add5.0mlof0.1NHCltoeachtubeandallowthetubestositforseveralhourstoelutethenucleotidesfromthepaper.

9. Packdownthepaperwithaglassrod(centrifugeinaclinicalcentrifugeifnecessary)andremoveanaliquotoftheliquidforspectrophotometricassay.

10. MeasuretheabsorbanceofeachofthefournucleotidesattheindicatedUVwavelength(havingfirstblankedtheinstrumentwith0.1NHCl).

    Base	Wavelength	MolarExtinctionCoefficient
    Guanine	250nm	10.6
    Adenine	260nm	13.0
    Cytidylicacid	280nm	19.95
    Uridylicacid	260nm	9.89

11. Usethemolarextinctioncoefficientstodeterminetheconcentrationofeachbaseinthesample.Calculatethepercentcompositionofeachbase,andthepurine/pyrimidineratio.

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