Yale RNA prep
来自 : 蚂蚁淘
YaleRNAprep(RebeccaD.Burdine,MichaelJ.Stern[modifiedbyS.M.Hettenbach])- Add8mlofTRIZOLto2mlpackedwormsin15mlcentrifugetube.Vortexandinverttubetosolubilizeandlysewormsforatleast10min.
- Divideintotwo15mlc/ftubes--5mleach.
- Invertforadditional5min.Solutionshouldbepinkandwillcontainthread-likematerial.Spinat4°C,4000rpmfor20mintoremoveinsolublematerial.Allspinsdoneinfixedanglerotorwithwhiteinserts.
- Transfersupernatanttonew15mlc/ftubes(2).Add800
lchloroformtoeachtube. - Vortex15sec,incubateatroomtemperaturefor3min.Smearyprepswillresultifvortexingnotlongenough.
- Spinat4°C,4000rpmfor20mintoseparatephases.
- Transferupperphasetonew15mlc/ftubes(2).Add2mlisopropanoltoeachtube.
- Inverttomix&incubateatRTfor10min.Spinat4°C,4000rpmfor30min.
- Removes/nwithpipetwithoutdisturbingthepellet.
- Add400ul75%EtOH,vortexbriefly.
- Spinat4°C,4000rpmfor10min.
- Removes/nwithpipetandinvertrackedtubesinhoodwithdoorhalfshut.Approx.10-15minutes;donotoverdry.
- Add300lDEPC-treatedddH2OtoeachtubeandresUSPendandcombinetubesusingwideopeningtips.Mayhavetoheatbrieflyat45-50°Ctocompletelydissolve.ReallygoodyieldsmayrequireaddingmoreH2O.
- Use2ltodoOD260andOD280todetermineconcentration.AnA260/280ratioof<1.6indicatespartiallydissolvedRNA.1OD260isequalto40g/mlofRNA.
- Storeat-80°Cin1mgaliquots,avoidrepeatfreezingandthawing.
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