0.(Optional)

BeforeusingOligotex-dT30,IrecommendtowashOligotex-dT30toremovefineparticleoflatex.Towashthelatex,transferappropriateamountofOligotex-dT30(300ulofthesUSPensionofOligotexper1mgoftotalRNA)intoamicrofugetube.Spinfor3minat12,000rpm.Discardthesupernatant.Gentlysuspendthelatexinthesamevolumeof10mMTris-HCl(pH7.5),1mMEDTA,0.1%SDS.Spinagain.Gentlysuspendthelatexinthesamebuffer.

1.Add1mgoftotalRNAdissolvedinRNase-freewaterto300uloftheOligotex-dT30suspension.

2.Incubatefor3minutesat65C.Chillonice.

3.Add0.2volumeof5MNaCl.Incubatefor10minutesat37C.

4.Centrifugefor3minutesat15,000rpm.Discardthesupernatant.

5.Suspendthepelletin1mlofwashingbuffer(10mMTris-HClpH7.5,1mMEDTA,0.5MNaCl,0.1%SDS).

6.Centrifugefor3minutesat15,000rpm.Discardthesupernatant.

7.Suspendthepelletin300ulofRNase-freewatercontaining0.1%SDS.

8.Incuabtefor5minutesat65C.Chillonice.

9.Centrifugefor3minutesat15,000rpm.Transferthesupernatantintonewmicrofugetube.

10.Carryoutphenol-extranction,chloroform-extractionandethanolprecipitationbystandardprocedure.Rinsethepelletwith75%ethanol.Dissolvethepoly(A)+RNAin10ulofRNase-freewater.

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