YaleRNAprep(RebeccaD.Burdine,MichaelJ.Stern[modifiedbyS.M.Hettenbach])

1. Add8mlofTRIZOLto2mlpackedwormsin15mlcentrifugetube.Vortexandinverttubetosolubilizeandlysewormsforatleast10min.
2. Divideintotwo15mlc/ftubes--5mleach.
3. Invertforadditional5min.Solutionshouldbepinkandwillcontainthread-likematerial.Spinat4°C,4000rpmfor20mintoremoveinsolublematerial.Allspinsdoneinfixedanglerotorwithwhiteinserts.
4. Transfersupernatanttonew15mlc/ftubes(2).Add800lchloroformtoeachtube.
5. Vortex15sec,incubateatroomtemperaturefor3min.Smearyprepswillresultifvortexingnotlongenough.
6. Spinat4°C,4000rpmfor20mintoseparatephases.
7. Transferupperphasetonew15mlc/ftubes(2).Add2mlisopropanoltoeachtube.
8. Inverttomix&incubateatRTfor10min.Spinat4°C,4000rpmfor30min.
9. Removes/nwithpipetwithoutdisturbingthepellet.
10. Add400ul75%EtOH,vortexbriefly.
11. Spinat4°C,4000rpmfor10min.
12. Removes/nwithpipetandinvertrackedtubesinhoodwithdoorhalfshut.Approx.10-15minutes;donotoverdry.
13. Add300lDEPC-treatedddH₂OtoeachtubeandresUSPendandcombinetubesusingwideopeningtips.Mayhavetoheatbrieflyat45-50°Ctocompletelydissolve.ReallygoodyieldsmayrequireaddingmoreH₂O.
14. Use2ltodoOD₂₆₀andOD₂₈₀todetermineconcentration.AnA260/280ratioof<1.6indicatespartiallydissolvedRNA.1OD₂₆₀isequalto40g/mlofRNA.
15. Storeat-80°Cin1mgaliquots,avoidrepeatfreezingandthawing.

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