ThepurityofthetransgeneDNAusedformicroinjectioniscriticalforthesuccessfulproductionoftransgenicfoundermice.DNAimpuritiesintheformofbacterialendotoxinsororganiccontaminantscandecreasetheintegrationefficiencyoftransgeneDNAortheviABIlityofmicroinjectedembryos. AtransgeneconstructpreparedfromgenomicDNAispreferentialtoonepreparedfromaCDNAclone.WhiletherearesomereportsofcDNAtransgeneexpressionintheliterature,intronsintheDNAincreasethelikelihoodoftransgeneexpression.Plasmidvectorsequencesshouldalsobecleavedawayfromthetransgeneconstructtobeusedformicroinjection.Thepresenceofplasmidsequencesinterfereswithtransgeneexpression.ListedbelowareguidelinesforthepreparationofDNAformicroinjection. UseplasmidDNAthathasbeenpurifiedonacesiumchloridegrADIenttoobtainDNAofhighestpurity.Digestapproximately20µgofplasmidDNAwitharestrictionenzymethatwillremovetheplasmidsequencesfromthetransgenicDNA. RuntheDNAonanagarosegelandelutethebandcontainingthetransgenicDNAtobeinjected.Mostelutionmethodsworkfine.WehaveusedGeneCleanfromBio101.TheQiaExgelextractionkitfromQiagenwillalsowork.Phenolfreemethodsthatleavelittlesaltcontaminationaredesirable. ResolubilizetheelutedDNAininjectionbuffer: 10mMTris,pH7.5 0.1mMEDTA,pH8.0 Whenpreparingtheinjectionbuffer,usedisposableplastic,freeoftracesofdetergents.Usereagentsdesignatedsolelyfortissuecultureworktoavoidtracecontaminants.Solutionsshouldbefilteredwitha0.2µmfiltertoremoveparticulateswhichmayclogtheinjectionneedle.ThepurityoftheDNAsampletobemicroinjectedmustbedocumentedbydigestingwithatleast2enzymesthatcutwithinthetransgeneandproducefragmentsofthepredictedsized. SubmitageltoverifythepurityanddetermineconcentrationoftheDNA.Supply1-5µgofDNAadjustedtoafinalconcentrationof100ng/µl.WewilldilutetheDNAto1-3µg/mlformicroinjection.ToohighaDNAconcentrationwillcausedevelopmentalarrestofembryos.Toolowaconcentrationdecreasesthenumberoftransgenicfounders.DNAconcentrationisbestdeterminedbypreparingserialdilutionsoflamBDaDNAofknownconcentration(25,50,100,200,400ngoflambdaDNA)andusingthemasstandards.RunthelambdaDNAsandtheDNAtobemicroinjectedonageltodeterminetheDNAconcentrationofthedilutedDNAformicroinjection.Wewillalsoverifytheconcentrationbyfluorimetry.