《trizol法提rna》doc版

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1、RNAsampletreatmentbeforeRealtime-PCRprotocolPart1.RNAExtraction(BaseonInvitrogenTRIZOL®Reagent,Cat.No.15596-026)Procedure1.HOMOGENIZATION1.1.Aspiratethemediumfromthewells.1.2.AddTRIZOL®Reagent1ml/well,andpassingthecelllysateseveraltimesthroughapipette.(Baseon6wellsplate)Note:Theamou

2、ntofTRIZOLReagentaddedisbasedontheareaoftheculturedish(1mlper10cm2)andnotonthenumberofcellspresent.AninsufficientamountofTRIZOLReagentmayresultincontaminationoftheisolatedRNAwithDNA.1.3.Transferthehomogenatesolutiontofreshtubes(1.5mlEppendorftube)2.PHASESEPARATION2.1.Incubatethehomo

3、genizedsamplesfor5minutesat15to30°Ctopermitthecompletedissociationofnucleoproteincomplexes.2.2.Add0.2mlofchloroformper1mlofTRIZOLReagent.Capsampletubessecurely.Shaketubesvigorouslybyhandfor15secondsandincubatethemat15to30°Cfor2to3minutes.2.3.Centrifugethesamplesatnomorethan12,000×gf

4、or15minutesat2to8°C.Note:Followingcentrifugation,themixtureseparatesintoalowerred,phenol-chloroformphase,aninterphase,andacolorlessupperaqueousphase.RNAremainsexclusivelyintheaqueousphase.3.RNAPRECIPITATION3.1.Transferthecolorlessupperaqueousphasetoafreshtube.(1.5mlEppendorftube)3.2

5、.Use0.5mlofisopropylalcohol(per1mlofTRIZOLReagentusedfortheinitialhomogenization).Incubatesamplesat15to30°Cfor10minutesandcentrifugeatnomorethan12,000×gfor10minutesat2to8°C.4.RNAWASHRemovethesupernatant.WashtheRNApelletoncewith1ml75%ethanol(usethenucleasefreewaterdilute),addingatlea

6、st1mlof75%ethanol(per1mlofTRIZOLReagentusedfortheinitialhomogenization).Mixthesamplebyvortexingandcentrifugeatnomorethan7,500×gfor5minutesat2to8°C.5.REDISSOLVINGTHERNA5.1.DrytheRNApellet(air-dry),sometimeneedtousetips,openthecapandlaydownthetube.5.2.DissolveRNAinRNase-freewater(90µl

7、),andincubatingfor10minutesat55°Cto60°C.5.3.Storedat-70°C.Part2.DNaseDigestionofRNAbeforeRNACleanup(BaseonQIAGENtheRNase-FreeDNaseSet,Cat.no.79254):Note:DonotvortexthereconstitutedDNaseI.DNaseIisespeciallysensitivetophysicaldenaturation.Mixingshouldonlybecarriedoutbygentlyinvertingt

8、hetube.Storeat–20°C