|
Author:ZuyuanQian |
Source:ContributedbyZuyuanQian |
Abstract:ThisisthestandardandcompleteNorthernblottingprotocolincludingpreparationofRNAgel,RNAsample,andprobe,hybridization,detectionandrecipesforsolutions |
|
Overview Theprotocolisdividedintothreesections: - ElectrophoresisofanRNApreparationunderdenaturingconditionsinanagarose-formaldehydegel
- TransferoftheRNAfromthegeltoanylonornitrocellulosemembranebyupwardcapillarytransfer
- HybridizationanalysisoftheRNAsequencesofinterestusingalabeledDNAorRNAprobe.
I.Agarose/FormaldehydeGelElectrophoresis Preparegel:Dissolve0.75gagarosein36mlwaterandcoolto60oCinawaterbath.Whentheflaskhascooledto60oC,placeinafumehoodandadd5mlof10xErunningbufferand9mlformaldehyde.Pourthegelandallowittoset.Removethecomb,placethegelinthegeltank,andaddsufficient1xErunningbuffertocovertoadepthof~1mm. Preparesample:AdjustthevolumeofeachRNAsampleto6µlwithwater,thenadd2.5x6µlfreshlypreparedsampledenaturationmix.Mixbyvortexing,microcentrifugebrieflytocollectliquid,andincubate15minat55oC.Coolonicefor2min,thenadd2µlloADIngdyemix. Rungel:Runthegelin1xErunningbufferat100voltfor10min,thenat65voltfor90min
II.TransferofRNAfromGeltoMembrane Preparegelfortransfer:PlacethegelinanRNase-freedishandrinsewithchangessufficientdeionizedwatertocoverthegelfor4x20min. TransferRNAfromgeltomembrane:
- Filltheglassdishwithenough20xSSPE.
- Cut2piecesofWhatman1MMpaper,placeitontheglassplateandwetitwith20xSSPE.
- Placethegelonthefilterpaperandsqueezeoutairbubblebyrollingaglasspipet.
- Cutfourstripsofplasticwrapandplaceovertheedgesofthegel.
- Cutapieceofnylonmembrane(MSI,Catalog#N00HY320F5)justlargeenoughtocoverthegelandwettedinwater.Placethewettedmembraneonthesurfaceofthegel.Trytoavoidgettingairbubblesunderthemembrane.
- Floodthesurfaceofthemembranewith20xSSPE.Cut5sheetsofwhatman3MMpapertothesamesizeasmembraneandplaceontopofthemembrane.
- Putpapertowelsontopofthewhatman3MMpapertoaheightof~6cm,andaddaweighttoholdeverythinginplace.
- Leaveovernight.
Preparemembraneforhybridization:Removepapertowelsandfilterpapersandrecoverthemembraneandflattenedgel.Markinpencilthepositionofthewellsonthemembraneandensurethattheup-downandback-frontorientationarerecognizable.Rinsethemembranein5xSSPE,thenplaceitonasheetofWhatman3MMpaperandallowtotry.PlaceRNA-side-downonaUVtransIlluminator(254nmwavelength),andirradiateforappropriatelengthoftime.
III.HybridizationAnalysis PrepareDNAorRNAprobe(>108dpm/µg): TheprobelabeledwithRidiprimerDNAlabellingsystem(AmershamLIFESCIENCE):
- DilutetheDNAtobelabelledtoaconcentrationof2.5-25ngin45µlofsterilewater.
- DenaturetheDNAsamplebyheatingto95-100oCfor5mininaboilingwaterbath.
- Centrifugebrieflytobringthecontentstothebottomofthetube,andputonicefor10min.
- AddthedenaturedDNAtothelabellingmixandreconstitutethemixbygentlyflickingthetubeuntilthebluecolourisevenlydistributed.
- Add5µlofRedivue[32P]dCTPandmixbygentlypipettingupanddown.Centrifugebrieflytobringthecontentstothebottomofthetube,thenincubateat37oCfor30min.
- TheprobeispurifiedusingProbeQuantTMG-50microcolumns(Amershampharmaciabiotech):
- G-50microcolumnpreparation.ResUSPendtheresininthecolumnbyvortexing,loosenthecapone-fourthturnandsnapoffthebottomclosure.Placethecolumnina1.5mlscrew-capmicrocentrifugetubeforsupport,thenpre-spinthecolumnfor1minat3000rpminanEppendorfmodel5415C.
- Placethecolumninanew1.5mltubeandslowlyapply50µlofthesampletothetop-centeroftheresin,beingcarfulnottodisturbtheresinbed.Spinthecolumnat3000for2min.Thepurifiedsampleiscollectedinthebottomofthesupporttube.
Hybridization:
- Pre-hybridization:Wetthemembraneinthe5xSSPEandplaceitRNA-side-upinahybridizationtubeandadd5mlpre-hybridizationsolution,thenplacethetubeinthehybridizationovenandincubatewithrotation6hrat42oCforDNAprobeor60oCforRNAprobe.
- Hybridization:Double-strandedprobewasdenaturedbyheatinginawaterbathfor10minat100oC,thentransfertoice.Pipetthedesiredvolumeofprobeintothehybridizationtubeandcontinuetoincubatewithrotationovernightat42oCforDNAprobeor60oCforRNAprobe.
Autoradiography:
- Themembranewaswashedtwicefor5-10minwithwash-bufferatroomtemperature,andtwicefor15minat65oC.forwithprewarmed(65oC)wash-buffer.
- Removefinalwashsolutionandrinsemembranein5xSSPEatroomtemperature.BlotexcessliquidandcoverinUV-transparentplasticwrap.Donotallowmembranetodryoutifitistobereprobed.
- Blotwasexposedat-80oCunsingKodakXARfilmandx-rayintensifyingscreens.
ReagentsandSolutions - 10xE:0.2MMOPS,0.05MNaAcand0.005MEDTA,adjusttopH7.0withNaOH;
- Sampledenaturationmix(100µl):64.6µlFormamide,22.6µlFormaldehydeand13µl10xE;
- Loadingdayemix:50%Glycerol,0.3%Xylenecyanol,0.3%bromophenolblueand1mMEDTA;
- 20xSSPE:3MNaCl,0.25MNaH2PO4and0.02MEDTA,adjusttopH7.4;
- Pre-hybridizationsolution:2.5mlformamide,1ml5xP,1.4mlH2O,0.292gNaCland0.1mlspermDNA;
- 5xP:0.25MTris-HClpH7.5,0.5%Sodiumpyrophosphate,1%Polyvinylpyrolidone,1%Bovineserumalbumine,1%Ficolland5%SDS;
- Wash-buffer:1%SDS+0.1%20xSSPE.
|
================ 蚂蚁淘在线 ================
免责声明:本文仅代表作者个人观点,与本网无关。其创作性以及文中陈述文字和内容未经本站证实,对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺,请读者仅作参考,并请自行核实相关内容
版权声明:未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的,应该授权范围内使用,并注明“来源:蚂蚁淘在线”。违反上述声明者,本网将追究其相关法律责任。
-
资质认证
获得国家资质,权威认证!
-
全国联保
全国联保,官方无忧售后
-
正规发票
正规发票,放心购买
-
签订合同
签订合同,保障您的权益
/**/