Joe mRNA prep 实验方法
来自 : 蚂蚁淘
Joe"smRNAprep(AudreyGasch,PatBrown[modifiedbyS.M.Hettenbach])Oligo-dTcelluloseprep
- DumpAmbionvialcontentsinto50mlc/ftube
- Add10ml1xNETStovial,cap,rinsevial,dump10mlintoc/ftube
- Spintube3000rpm,2minat4°Cinswingingbucketwithnobrake,removes/n
- Add10ml1xNETStotube,resUSPendcontentsbyswirling
- Spintube3000rpm,2minat4°Cinswingingbucketwithnobrake,removes/n
- Repeatsteps4and5twomoretimesforatotalof4washes
- Add10ml2xNETSandstoreat4°C
- Prepcellulosethedaybeforeyouneedit
mRNAprep
- Dilute1mgtotalRNAto1mlwith10mMTrispH7.4inc/ftube
- Ina2mlBioRadminicolumn(#731-1550)sealedonbottom,mix1mlresuspendedresinwith1mlof1mg/mltotalRNAfromstep1.CapandParafilmcolumn,tapetorocker,andbindatRTfor1houron5.5setting
- Breakbottomoffcolumnandplacein16x125mmtesttubeandallowcontentstoflowthrough
- Washresinfivetimeswith0.7ml1xNETS,slowflowisnormal
- Transfercolumntosupportandelute2xwith0.7ml10mMTrispH7.4preheatedto70°Cintoseparate1.7mltubes.Elutewith150
lofthe0.7mlvolumeatatimekeepingrestat70°C. - Add0.7mlchloroformtoeachtubetoremoveresiduecellulose,mixwell,spin13,000rpmfor1minatRT,transferupperphasetonewtube
- Precipitatewith0.1volume3MNaOAcpH7.0and1volisopropanolat-20°Cfor1hour
- Spin13,000rpmfor30minat4°C
- Removes/nandairdry
- Resuspendbothtubesinfinalvolumeof20lofDEPCddH2O
- SpecOD260&OD280on1lmRNA.1OD260isequalto40g/mlofRNA
- Storeat-80°C,avoidrepeatfreezingandthawing
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