97promega_SV_Total_RNA_Isolation_System说明书

TechnicalManualSVTotalRNAIsolationSystemINSTRUCTIONSFORUSEOFPRODUCTSZ3100,Z3101ANDZ3105.PRINTEDINUSA.Revised12/06Part#TM048AF9TM0481206TM048tm048.1206.qxp1/9/200711:02AMPage1PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone[phone]·Fax[phone]·(≤30mg)......................10C.PreparationofLysatesfromTissueSamples30mg....................................12D.LysisofCulturedCells......................................................................................12E.RNAPurificationbyCentrifugation(Spin)....................................................13F.RNAPurificationbyVacuum..........................................................................15V.DeterminationofRNAYieldandQuality.................................................17VI.Troubleshooting...............................................................................................19VII.References.........................................................................................................23VIII.Appendix...........................................................................................................23A.IsolationofTotalRNAfromLeukocytes........................................................23B.IsolationofTotalRNAfromPlantTissue......................................................25C.IsolationofRNAfromGram-Positive(B.subtilis)andGram-Negative(E.coli)Bacteria..............................................................25D.IsolationofRNAfromYeast.............................................................................26E.FurtherTipsonPreparingLysates..................................................................26F.AdherentCultureCells......................................................................................27G.CompositionofBuffersandSolutions............................................................28H.RelatedProducts.................................................................................................28I.DescriptionThepurityandintegrityofRNAisolatedfromtissueorculturedcellsarecriticalforitseffectiveuseinapplicationssuchasreversetranscriptionPCR(RT-PCR),real-timePCR,RNaseprotectionassays,Northernblotanalysis,oligo(dT)selectionofpoly(A)+RNA,invitrotranslationandmicroarraySVTotalRNAIsolationSystemAlltechnicalliteratureisavailableontheInternetat::[e-mail]1/9/200711:02AMPage1analysis.Inrecentyears,RT-PCRhasemergedasapowerfulmethodtoidentifyandquantitatespecificmRNAsfromsmallamountsoftotalRNAandmRNA.Astheuseofamplificationasaresearchtoolhasgrown,theneedformethodstorapidlyisolatehigh-qualityRNA,substantiallyfreeofgenomicDNAcontamination,fromsmallamountsofstartingmaterial(i.e.,tissueandculturedcells)hasalsoincreased.TheSVTotalRNAIsolationSystem(a)hasbeendesignedtoaddresstheseneeds.TheSVTotalRNAIsolationSystemprovidesafastandsimpletechniqueforpreparingpurifiedandintacttotalRNAfromtissues,culturedcellsandwhitebloodcells(seeSectionVIII.A)inaslittleasonehour,dependingonthenumberofsamplestobeprocessed.LiketheWizard®PlusSVMiniprepsDNAPurificationSystem(Cat.#A1330),eitheraspinorvacuum(“SV”)purificationprotocolcanbeused.Upto60mgoftissuecanbeprocessedperpurification,dependingonthetype,functionandRNAexpressionlevelsofthetissue.ThesystemalsoincorporatesaDNasetreatmentstepthatisdesignedtosubstantiallyreducegenomicDNAcontamination,whichcaninterferewithamplification-basedmethodologies.Purificationisachievedwithouttheuseofphenol:chloroformextractionsorethanolprecipitations,andthereisnoDNasecarryoverinthefinalRNApreparation.TheSVTotalRNAIsolationSystemcanalsobeusedtoisolatebothgenomicDNAandRNAfromthesamesample.ForprotocolsandadditionalinformationontheuseofthissystemforDNAisolation,pleaseseereference1orvisitthePromegawebsiteat: