Joe"smRNAprep(AudreyGasch,PatBrown[modifiedbyS.M.Hettenbach])

Oligo-dTcelluloseprep

1. DumpAmbionvialcontentsinto50mlc/ftube
2. Add10ml1xNETStovial,cap,rinsevial,dump10mlintoc/ftube
3. Spintube3000rpm,2minat4°Cinswingingbucketwithnobrake,removes/n
4. Add10ml1xNETStotube,resUSPendcontentsbyswirling
5. Spintube3000rpm,2minat4°Cinswingingbucketwithnobrake,removes/n
6. Repeatsteps4and5twomoretimesforatotalof4washes
7. Add10ml2xNETSandstoreat4°C
8. Prepcellulosethedaybeforeyouneedit

mRNAprep

1. Dilute1mgtotalRNAto1mlwith10mMTrispH7.4inc/ftube
2. Ina2mlBioRadminicolumn(#731-1550)sealedonbottom,mix1mlresuspendedresinwith1mlof1mg/mltotalRNAfromstep1.CapandParafilmcolumn,tapetorocker,andbindatRTfor1houron5.5setting
3. Breakbottomoffcolumnandplacein16x125mmtesttubeandallowcontentstoflowthrough
4. Washresinfivetimeswith0.7ml1xNETS,slowflowisnormal
5. Transfercolumntosupportandelute2xwith0.7ml10mMTrispH7.4preheatedto70°Cintoseparate1.7mltubes.Elutewith150lofthe0.7mlvolumeatatimekeepingrestat70°C.
6. Add0.7mlchloroformtoeachtubetoremoveresiduecellulose,mixwell,spin13,000rpmfor1minatRT,transferupperphasetonewtube
7. Precipitatewith0.1volume3MNaOAcpH7.0and1volisopropanolat-20°Cfor1hour
8. Spin13,000rpmfor30minat4°C
9. Removes/nandairdry
10. Resuspendbothtubesinfinalvolumeof20lofDEPCddH₂O
11. SpecOD₂₆₀&OD₂₈₀on1lmRNA.1OD₂₆₀isequalto40g/mlofRNA
12. Storeat-80°C,avoidrepeatfreezingandthawing

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