Purification of Gene Targeting Vector DNA for Electroporation
PurificationofGeneTargetingVectorDNAforElectroporation
1.Purifyplasmidfrombacteria.
WerecommendtheQiagenEndoFreePlasmidMaxikitforthepurificationofthetargetingvectorplasmidfrombacteria.Pleasefollowthedirectionsinthekit.ElectroporationofQiagenpurifiedDNAhasbeenusedsuccessfullybyanumberoflabs.Alternatively,plasmidDNAcanbepurifiedbyCsClbanding.2.Linearize200microgramsofplasmidDNAwiththeappropriaterestrictionenzymedigest.
RunaDNAonaminigeltoverifythatdigestioniscomplete.ExtracttheDNAwithphenol-chloroform,thenwithchloroformandprecipitatebyaddingNaClandethanol.MakesureyouusefreshphenolwithneutralpHformaximumDNArecoveryandhighestcellviABIlityinelectroporation.WashtheDNApelletin70%ethanolandallowtoitairdry.ResUSPendtheDNAinsterileTE(10mMTris-HCl,pH8.0,1.0mMEDTA)at2mg/mlanddeliverittotheTransgenicCoreforelectroporation.Priortoelectroporation,wewillverifytheconcentrationandrunitonaminigeltocheckthesizeandlookfordegradation.
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