1.Transfer5-10μgofpoly(A)+RNAtoasterilemicrofugetube.Adjustthevolumeofthesolutionto40μlwithRNase-freeH2O.Heattheclosedtubeto70°Cfor5minutes,andthenquicklytransferthetubetoanicewaterbath.

2.Tothechilledmicrofugetube,add:

0.1Mdithiothreitol2.5μl

placentalRNaseinhibitor200units

oligo(dT)12-1810μl

10xreversetranscriptasebuffer25μl

20mMsolutionofdGTP,dATP,anddTTP10μl

125μMdCTP10μl

10mCi/ml[-32P]dCTP(sp.act.>3000Ci/mmole)100μl

RNase-freeH2Oto240μl

reversetranscriptase(2000units)10μl

IMPORTANTAddthereversetranscriptaselast.

Mixthecomponentsbygentlytappingthesideofthetube.Collectthereactionmixtureinthebottomofthetubebybriefcentrifugationinamicrofuge.Incubatethereactionfor1hourat45°C.

Asanalternative,[-32P]dCTPofspecificactivity800Ci/mmolecanbesubstitutedinthisreaction.Ifthissubstitutionismade,thenomitthe125μMdCTPfromthereactionmixture.

3.Stopthereactionbyadding:

0.5MEDTA(pH8.0)10μl

10%(w/v)SDS10μl

Mixthereagentsinthetubewell.

4.Add30μlof3NNaOHtothereactiontube.Incubatethemixturefor30minutesat68°CtohydrolyzetheRNA.

5.Coolthemixturetoroomtemperature.Neutralizethesolutionbyadding100μlof1MTris-Cl(pH7.4),mixingwell,andthenadding30μlof2.5NHCl.CheckthepHofthesolutionbyspotting<1μlonpHpaper.

6.PurifytheCDNAbyextractionwithphenol:chloroform.

7.MeasuretheproportionofrADIolabeleddNTPsthateitherareincorporatedintomaterialprecipitatedbyTCAoradheretoaDE-81filter(pleaseseeAppendix8intheprintversionofthemanual).CalculatetheyieldofcDNAasfollows:

Inareactioncontaining1.5nmolesofthelimitingdNTP:cpmincorporatedx1.5nmolesdCTPx330ng/nmolex160=ngofcDNAsynthesizedtotalcpm

8.SeparatetheradiolabeledprobefromtheunincorporateddNTPsbychromatographythrougha5-mlcolumnof

SephadexG-50.

IMPORTANTPerformthisstepandallsubsequentstepswithsiliconizedtubes.

9.TotheradiolabeledcDNA,addtenfoldexcessbyweightofthedriverRNAthatwillbeusedtosubtractthecDNAprobe,0.2volumeof10Mammoniumacetate,and2.5volumesofice-coldethanol.Incubatethemixturefor10-15minutesat0°C,andthenrecoverthenucleicacidsbycentrifugationatmaximumspeedfor5minutesat4°Cinamicrofuge.

10.Removealloftheethanolbyaspiration,andstoretheopentubeonthebenchforafewminutes.Dissolvethenucleic

acidsin6μlofRNase-freeH2O.

11.Tothedissolvednucleicacids,add:

2Msodiumphosphate(pH6.8)2μl

SDS/EDTAsolution2μl

12.Eithercoverthesolutionwithadropoflightmineraloilordrawthemixtureintoasiliconized,disposable20-μlglasscapillarytubeandsealtheendsofthetubeintheflameofaBunsenburner.

13.Placethemicrofugetubeorsealedcapillarytubeinaboilingwaterbathfor5minutes.Transfertoawaterbathsetat68°C,andallowthenucleicacidstohybridizetoCrot=1000molesseconds/liter.Tocalculatethetimerequiredtoreachvalue,pleaseseepage9.44oftheprintversionofthemanual.

14.Removethemicrofugetubeorcapillarytubefromthewaterbath.Useadrawn-outPipettetipattachedtoamicropipettortoremovethehybridizationsolutionfromthemicrofugetube,oropentheendsofthecapillarytubewithafileordiamondpen.Transferthehybridizationmixtureintoatubecontaining1mlofSPSbuffer.

15.Separatethesingle-strandedanddouble-strandednucleicacidsbychromatographyonhydroxyapatiteat60°C.

Measuretheamountofradioactivityineachfractionbyliquidscintillationcounting.Atleast90%oftheinput[32P]cDNAshouldhavehybridizedtothemRNAandbepresentinthe>0.36Msodiumphosphatewash.

16.Poolthefractionscontainingthesingle-strandedcDNAandconcentratethembyrepeatedextractionswithisobutanolextraction:Addanequalvolumeofisobutanol.Mixthetwophasesbyvortexing,andcentrifugethemixtureatmaximumspeedfor2minutesatroomtemperatureinamicrofuge.Discardtheupper(organic)phase.Repeattheextractionwithisobutanoluntilthevolumeoftheaqueousphaseis<100μl.

17.RemovesaltsfromthecDNAbyspun-columnchromatographythroughSephadexG-50equilibratedinTE(pH8.0)

containing0.1%SDS.

IMPORTANTDonotuseethanolprecipitationtoconcentratethecDNAasthepresenceofphosphateionsinterfereswithprecipitation.Donotusedialysistoremovephosphateions,asthecDNAwillsticktothedialysisbag.

18.MeasuretheamountofradioactivityinthesampleandcalculatetheweightofDNAinthesubtractedprobe.

19.RepeatSteps9-18.

Between10%and30%ofthecDNAwillformhybridswiththedriverRNAduringthesecondroundofhybridization.

ItisnotnecessarytoconcentrateorremovesaltsfromthefinalpreparationofcDNAifitistobeusedtoprobeacDNAlibrary.TheradiolabeledcDNAcanbeusedforhybridizationwithoutdenaturation.Thesubtractivehybridizationsshouldbecarriedoutasrapidlyaspracticable,andtheprobeshouldbeusedwithoutdelay.Use5x107dpmofradiolabeledcDNAforeach150-mmfilterand5x106to1x107dpmforeach90-mmfilter.

IfagenomicDNAlibraryisscreenedwiththeradiolabeledsubtractedprobe,oligo(dA)canbeaddedtotheprehybridizationandhybridizationreactionsat1μg/mltopreventnonspecifichybridizationbetweentheoligo(dT)tails

ofthecDNAandoligo(dA)tractsinthegenomicDNA.

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