DIG-RNA-LABELING-KIT(SP6T7)

(SP6/T7)RNAlabelingwithdigoxigenin-UTPbyinvitrotranscriptionwithSP6andT7RNApolymeraseCat.No.11175025910Kitfor2×10labelingreactionsVersionApril2010Storeat15to25°C1.WhatthisProductDoesNumberofTests1kitissufficientfor2×10labelingreactions.KitContentsBottleLabelContentsIncludingFunction1pSPT18DNA�40l(0.25mg/ml)�Cloning/transcriptionvector;subclonesaretranscribedintoRNAprobesbyT7orSP6RNApolymerase�AccessionNo.pSPT18DNAsequenceisA133882pSPT19DNA�40l�[0.25mg/ml]�Cloning/transcriptionvector;subclonesaretranscribedintoRNAprobesbyT7orSP6RNApolymerase3ControlDNA1pSPT18-Neo�20l�[0.25mg/ml]cleavedwithPvuII�TranscriptionofcontrolDNA1byT7RNApolymeraseaccordingtothestandardpro-tocolproducesDIG-labeled“antisense”Neotranscriptsof760basesinlength.�Useascontrolinlabelingandhybridizationreactions�containsDNAfragmentsof798and3281bpduetocleavageatthe2PvuIIsitesontheplasmid4ControlDNA2pSPT19-Neo�20l�[0.25mg/ml]cleavedwithPvuII�TranscriptionofcontrolDNA2bySP6RNApolymeraseaccordingtothestan-dardprotocolproducesDIG-labeled“anti-sense”Neotranscriptsof760basesinlength.�Useascontrolinlabelingandhybridizationreactions�containsDNAfragmentsof316and3761bpduetocleavageatthe2PvuIIsitesontheplasmid(oneatpos.761withintheneogene)5LabeledcontrolRNA*�100l�(100ng/l)�DIG-labeled“antisense”NeoRNA(7)(760bases),transcribedwithT7RNApoly-merasefrom4l(equivalentto1g)PvuII-linearizedpSPT18-NeoDNA(vial3)accordingtothestandardprotocol.ThetranscriptionassaywasnottreatedwithDNaseI.Afterethanolprecipitation,itwasdissolvedindimethylpyrocarbonate-treatedwater.�Thesolutioncontainsapprox.10gofDIG-labeledNeoRNAand1gpSPT18-NeotemplateDNA�Forsemi-quantitativeestimationofDIG-labeledRNAanduseasahybridizationcontrol�containsDNAfragmentsof798and3281bp.6Unlabeledcon-trolRNA�20l,unlabeledcontrolRNA,�[200g/ml]�UnlabeledNeopoly(A)“sense”RNAindimethylpyrocarbonate-treatedwater.�TheNeopoly(A)RNAissynthesizedbyinvitrotranscriptionandis1kblong�TargetRNAtopracticeRNA/RNAhybrid-izations;whenappliedtoamembrane,thisRNAwillhybridizewiththelabeledcontrolRNA(vial5)7NTPlabelingmixture*�40lnucleotidemixture,�10×,[10mMATP,10mMCTP,10mMGTP,6.5mMUTP,3.5mMDIG-11-UTP,pH7.5(20oC)]8Transcriptionbuffer�40l�10×conc.9DNaseI,RNase-free*�40l�[10U/l]�DegradesDNAtemplateafterthelabelingreaction10ProtectorRNaseInhibitor*�20l�[20U/l]�PreventsthedegradationofRNAduringthelabelingreaction11SP6RNAPolymerase*�20l�[20U/l]�SynthesizesRNAfromaDNAtemplate12T7RNAPolymerase*�20l�[20U/l]�SynthesizesRNAfromaDNAtemplateBottleLabelContentsIncludingFunction0410.11244175001*availablefromRocheAppliedScience215to25°Cthroughtheexpirationdateprintedonthelabel.Thekitisshippedondryice.AdditionalEquipment/ReagentsRequiredInadditiontothereagentslistedabove,youmustprepareseveralsolutions.Inthetableyouwillfindanoverviewoftheequipmentandreagentsneededforthedifferentprocedures.Detailedinformationisgivenatthebeginningofeachprocedure.ApplicationsDIG-labeledRNAisusedforhybridizationto�Northernblots�Southernblots�insituhybridization�plaqueorcolonylifts�RNaseprotectionexperimentsLAsthelinkagebetweenDIGandUTPisresistanttoalkali,DIG-labeledRNAcanbefragmentedbyalkalinetreatment.SlightlyreducingthesizeoftheDIG-labeledRNAprobemaymakeitmoresuitableforcertainapplicationsininsituhybridization(5,6).2.HowtoUsethisProduct2.1BeforeYouBeginGeneralHandlingRecommendationsThistablegivessomegeneraltipsforsuccessfulDIGlabelinganddetection.SampleMaterial�linearizedplasmidDNA�PCRproductProcedureEquipmentReagents2.2DIGRNAlabelingwaterbath�H2O,sterile,doubledistilledwater*treatedwithDMPC�EDTA,0.2M,pH8.02.3Determina-tionoflabelingefficiency�nylonmembranespositivelycharged*�UV-transillumi-natoror�UVcross-linker�RNAdilutionbuffer�DIGWashandBlockBufferSet*�Anti-digoxigenin-AP*�CSPD*orCDP-Star*�DIGLuminescentDetectionKit*RecommendationGuidelineWorkundercleanconditions�AutoclaveDIGSystemsolutions�Filter-sterilizesolutionscontainingSDS�DonotaddTween20tosolutionsuntilaftertheyhavebeensterilizedUsecleanincubationtrays�Rigorouslycleanandrinselaboratorytraysbeforeeachuse�Whenperformingnorthernblotsusesteril-izedglasstraysandsolutionsforallwashinganddetectionsteps.Membranehandlingrequirements�Wearpowder-freegloves�HandlemembraneonlyontheedgesandwithcleanforcepsFeaturesoftheTemplateDNAThefollowingtableliststherecommendedfeaturesofthetemplate.SamplePreparation�LinearizetheDNAtemplatebycuttingatarestrictionenzymesitedownstreamfromtheclonedinsert.�Toavoidtranscriptionofundesirablesequences,usearestrictionenzymethatcreates5´overhangs.�Afterrestrictiondigest,purifytheDNAwiththeHighPurePCRProductPurificationKit*orviaphenol/chloroformextraction,andsubsequentethanolprecipitation.PreparationofWorkingSolutionsThistablelistscomposition,storageanduseoftheotherreagentsyouwillneedinadditiontokitcomponents.ControlReactionsForcheckingiftheDIGlabeledRNAprobehasbeenlabeledef