Protocol for Southern Blot using DIG from Jing Ding -Clara

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1、ProtocolforSouthernBlot(withDIGHighPrimerDNALabelingandDetectionStarterKitI)1.PurificationofTemplate1.5h–overnight2.Labeling1.3h–overnight2.1Add16μl(300ng-3μg)targetDNAtoatube.2.2Heatthesampleinaboilingwaterbathfor10min.2.3Quicklychillthesampleinanice/water

2、bath.2.4MixDIG-HighPrimethoroughly.2.5Add4μlmixedDIG-HighPrimetothedenaturedsample.2.6Centrifugebriefly.2.7Incubatesampleforatleast1hat37°C.Note:Ifyouhavealimitedamountoftemplate,youmaycontinuetheincubationovernight(upto20h)toincreasetheyieldoflabeledprobe.

3、2.8Add2μl0.2MEDTA(pH8.0)tothesample,and/orheatthesampleto65°Cfor10mintostopthereaction.3.DeterminationofProbeYield2–2.5h3.1Preparea1ng/μlworkingsolutionofthelabeledDNAprobewithDNADilutionBuffer,eg.,makea1:20dilution(1μllabeledproduct+19μlDNADilutionBuffer)f

4、or20ng/μlexpectedyieldofprobe(estimatedfrom1hlabelingof300ngDNA).3.2Preparea1ng/μlworkingsolutionofDIG-labeledControlDNA(originalconcentration=5ng/μl)inDNADilutionBuffer.3.3UsingtheDNADilutionBuffer,prepareseparateserialdilutionsofthelabeledprobeandthecontr

5、olDNA,asshowninthetablebelow:TubeDNA(μl)FromTube#DNADilutionBuffer(μl)OverallDilution(fromTubeD1)FinalConcentrationD1*None1ng/μlD22D11981:10010pg/μlD315D2351:3303pg/μlD45D2451:10001pg/μlD55D3451:33000.3pg/μlD65D4451:1040.1pg/μlD75D5451:330000.03pg/μlD85D645

6、1:1050.01pg/μlD90-50-0*WorkingsolutionoflabeledprobeorcontrolDNAfromStep3.1orStep3.2.3.4Prepareanarrowstrip(approx.3x5cm)ofPositivelyChargedNylonMembrane.3.5Apply1μlspotsfromprobedilutionsD2–D9ontheMembrane.3.6Inarowparalleltotheprobedilutions,apply1μlspots

7、ofthecorrespondingcontroldilutions.3.7Marklocationofeachprobeandcontrolspotwithapencil.3.1CrosslinkwithStratalinker120mJtofixthenucleicacidspotstothemembrane.3.2Transferthemembranetoaplasticcontainer(e.g.,apetridish)containing20mlWashingBuffer.3.3Incubatefo

8、r2minwithshaking.3.4DiscardtheWashingBuffer.3.5Incubatemembranefor30minin10mlBlockingSolution.3.6DiscardtheBlockingSolution.3.7Incubatethemembranefor30minin10mlAntibodySolution.3.8Washmembranetwice(2x1