生理所黄阿敏

Southern NorthernandWesternblotting 生理所黃阿敏 ComparisonofSouthern Northern andWesternanalysesofGeneX Southernhybridization FirstdescribedbyE M Southernin1975 ApplicationsofSouthernhybridizationRFLP s VNTR sandDNAfingerprintingCheckingofthegeneknockoutmiceTheflowchartofSouthernhybridization Southernhybridization Transferbuffer DetectionofanRFLPbySouthernblotting Detectionofthesickle cellglobingenebySouthernblotting Checkingofthegeneknockoutmice FlowchartofSouthernhybridization PreparingthesamplesandrunningthegelSoutherntransferProbepreparationPrehybridizationHybridizationPost hybridizationwashingSignaldetection IsotopeNon isotope Preparingthesamplesandrunningthegel Digest10pgto10 gofdesiredDNAsamplestocompletion Prepareanagarosegel loadsamples remembermarker andelectrophorese Staingelethidiumbromidesolution 0 5 g ml Photographgel withruler Criticalparameters I NotethecomplexityofDNAGenomicDNAAsingle copyofmammaliangene 3Kbaverageinlength10mgx3Kb 3x106Kb 10mgx1 106 10pgPlasmidDNAorPCRproducts0 1mgofa3KbplasmidDNA 100ng Geltreatment Acidtreatment0 2NHClsolutionDenaturationNaOHsolutionNeutralizationTris Clbuffer pH8 0 Southerntransfer Measuregelandsetuptransferassembly Wickintraywith20 xSSCGelNitrocelluloseorNylonfilters soakedinH2Oand20 xSSC 3MMWhatmanfilterpaperPapertowelsWeight AfterSoutherntransfer Dissembletransferpyramidandrinsenitrocellulosein2xSSCBakenitrocelluloseat80 Cfor2hrorUV crosslinkNylonmembraneforseconds Preparationofprobes Synthesisofuniformlylabeleddouble strandedDNAprobesPreparationofsingle strandedprobesLabelingthe5 and3 terminiofDNA Synthesisofdouble strandedDNAprobes NicktranslationofDNALabeledDNAprobesusingrandomoligonucleotideprimers Nicktranslation Preparationofsingle strandedprobes Synthesisofsingle strandedDNAprobesusingbacteriophageM13vectors SynthesisofRNAprobesbyinvitrotranscriptionbybacteriophageDNA dependentRNApolymerase Invitrotranscription Labelingthe3 terminiofdouble strandedDNAusingtheKlenowfragmentofE coliDNApolymeraseI lackof5 3 exonucleaseactivity Labelingthe3 terminiofdouble strandedDNAusingbacteriophageT4DNApolymerase Labelingthe5 terminiofDNAwithbacteriophageT4polynucleotidekinase Labelingthe5 and3 terminiofDNA T4polynucleotidekinaseactivity Non isotopelabeling Digoxigenin 11 dUTP DIG dUTP labelingDNAlabelingOligonucleotidelabelingRNAlabeling PCRLabeling RandomPrimedLabeling andRNALabeling Prehybridization Addprehybridizationsolutionandprehybridizeathybridizationtemperaturefor2 4hr Hybridization RemoveprehybridizationsolutionandaddhybridizationsolutionAdd500 000cpmoftheprobe mlhybridizationsolution Hybridizeovernightatappropriatetemperature Post hybridizationwashing Washtwice 15mineach in1xSSC 0 1 SDSatroomtemperature Washtwice 15mineach in0 25xSSC 0 1 SDSathybridizationtemp Criticalparameters II HomologybetweentheprobeandthesequencesbeingdetectedTm 81 16 6 logCi 0 4 G C 0 6 formamide 600 n 1 5 mismatch Factorscanbechanged Hybridizationtemp Washingtemp SaltconcentrationduringwashingHightemp lowsalt highstringencyLowtemp highsalt lowstringencyIf50 formamideisused42oCfor95 100 homology37oCfor90 95 homology32oCfor85 90 homology Comparisonofnitrocelluloseandnylonmembranes Signalsdetection AutoradioragraphyNon isotopedetectionsystemChemiluminescentdetectionColorimetricdetectionMulticolordetection Autoradiography Exposuretox rayfilm NorthernblottingorNorthernhybridization TechniquefordetectingspecificRNAsseparatedbyelectrophoresisbyhybridizationtoalabeledDNAprobe TheflowchartofNorthernhybridization PrepareRNAsamplesandrunRNAgelNortherntransferProbepreparationPrehybridizationHybridizationPost hybridizationwashingSignaldetection IsotopeNon isotope Preparationofagarose formaldehydegel E g Preparea350ml1 2 agarose formaldehydegel4 2gagarosein304 5gwater Microwave thencoolto60 C Add35ml10 xMOPSrunningbufferand10 5ml37 formaldehyde PreparationofRNAsamples Prepareapremix 5 lof10 xMOPSrunningbuffer8 75 lof37 formaldehyde25 lofformamide PrepareRNAsamples 38 75 lofpremixRNA 0 5to10 g waterto50 l IfthemRNAspeciesofinterestmakesuparelativelyhighpercentageofthemRNAinthecell 0 05 ofthemessage totalcellularRNAcanbeused IfthemRNAspeciesofinterestisrelativelyrare however itisadvisabletousepoly A RNA Incubate15minat55 C RunningtheRNAgel Add10 lformaldehydeloadingbuffertoeachsampleandloadgel Rungelat100to120Vfor 3hr Removegelfromtherunningtankandrinseseveraltimesinwater Placegelin10 xSSCfor45min Donotneedpost transferringgeltreatment AnexampleofNorthernblotting Northernblot RNAgel 28S 18S Westernblotting orimmunoblotting Techniquefordetectingspecificproteinsseparatedbyelectrophoresisbyuseoflabeledantibodies FlowchartofWesternblotting ElectrophoresingtheproteinsampleAssemblingtheWesternblotsandwichTransferringproteinsfromgeltonitrocellulosepaperStainingoftransferredproteinsBlockingnonspecificantibodysitesonthenitrocellulosepaperProbingelectroblottedproteinswithprimaryantibodyWashingawaynonspecificallyboundprimaryantibodyDetectingboundantibodybyhorseradishperoxidase anti Igconjugateandformationofadiaminobenzidine DAB precipitatePhotogr