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Purification of Gene Targeting Vector DNA for Electroporation

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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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Purification&nbsp;of&nbsp;Gene&nbsp;Targeting&nbsp;Vector&nbsp;DNA&nbsp;for&nbsp;Electroporation

PurificationofGeneTargetingVectorDNAforElectroporation

1.Purifyplasmidfrombacteria.

2.Linearize200microgramsofplasmidDNAwiththeappropriaterestrictionenzymedigest.

Purification of Gene Targeting Vector DNA for Electroporation