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SouthernBlottingDNAFingerprinting.ppt

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SouthernBlottingDNAFingerprintingSouthernBlotSouthernBlotToday’slaboratory:DNAfingerprintinginahypotheticalpaternitydetermination.Inthishypotheticalcase,DNAwasextractedfromsamplesobtainedfromtwopossiblefathers,amotherandchild.TheDNAwaswerecleavedwitharestrictionenzyme.LoadingGelsHowtohandleyourgelafterelectrophoresis…LoadGelsandthenwewilldiscussrecombinantDNAtechnologySampleVolumeAdd40ulperwellMUSTWARM5min.at65CDepurinationStepDenaturationStepGeneralSchematicforSouthernBlot:DNASettinguptheSouthernBlot:pg.3-82SettingupSouthernBlot:pg.3-82SettinguptheSouthernBlottransferOvernightthessDNAwilldiffusebycapillarytransferfromgelontonylonmembrane!Nextweekwewillstainthismembraneforsimilaritiesbetweenchildandparents.SouthernBlottingDNAFingerprintingAnalysisofSouthernBlotRemembertheSouthernBlotrequires…SouthernBlotrequiresthat…YouwillprobetheblottodayYouwillprobetheblottodayNext:manyrinsingstepsColorDevelopmenthttp://vector.cshl.org/resources/dnadetective.htmlAgarosegelelectrophoresisBiotin-DNAbindstoavidinlabelledalkalinephosphatase.Substrate:5-Bromo-4-chloro-3indolylphosphpate(BCIP)Product:NitroBlueTetrazoliumtobereducedtoaninsolublevisibleproduct.SouthernBlotAnalysis