MicroinjectionDNAPurification WehavefoundtheNucleoSpinTMExtractKit(availablefromClontech,CatalognumberK3051-1)isasimpleandfastwaytoobtainmicroinjectionqualityDNA.ThisisnottoexcludeothermethodsofDNApurificationbuttosaythatwefindthisisaconvenientmethodthatislessonerousthanCsClgrADIents. 1.Performrestrictiondigesttoliberatetransgenefromplasmidvectorsequences.Finalyieldshouldbe10-20microgramsoftransgeneinsert. 2.SeparaterestrictiondigestproductsonagarosegelusingeitherTBEorTAE.Useeitherlow-orstandard-meltingtemperatureagarose. 3.PlacegelontransIlluminator.Cutoutband(s)ofinterest.Useacleanrazorbladeorscalpel.RemoveasmuchexcessagaroseaspossIBLe.MinimizeDNAexposuretoUVlighttopreventphotochemicaldamage(lessthan1minute). 4.Transferagaroseslice(s)toapreweighedtube.Reweightubetodetermineweightofagaroseintube. 5.Foreach100mgofgel,add300microlitersbufferNT1.Ifagaroseconcentrationisgreaterthan1%,addproportionatelymorebuffer.Forexample,ifa2%agarosegelisused,add600microlitersbufferNT1foreach100mgofgel. 6.Dissolveat50degreesCentigradefor10minutes,vigorouslyvortexingevery2to3minutes. 7.PlaceaNucleoSpinTMcartridgeina2mlmicrotubeandload750microlitersdissolvedgelsliceontothecartridge.Spinatmaximumspeedfor60secondsinamicrocentrifuge.Discardtheflowthrough.Thecartridgehasacapacityof2microgramsDNA,soyoucanrunseveral750microlitersloadsofdissolvedgelslicethroughasinglecartridge. 8.Add750microlitersofbufferNT3tothecartridgeandspinatmaximumspeedfor60secondsinamicrocentrifuge.Discardtheflowthrough. 9.Replacetubewithafreshmicrotube.Spintheemptycartridgeatmaximumspeedfor60secondsinamicrocentrifugetocompletelyremovebufferNT3. 10.ElutetheDNAfromthecartridge:Replacetubewithafreshmicrotube.Add50microlitersofpreheated(60degreesCentigrade)elutionbuffertocartridge.Spinatmaximumspeedfor60secondsinamicrocentrifuge.Elutionbuffer:10mMTris-HCl,pH8.5,0.02micronfiltered.CheckthepHoftheelutionbeforejustbeforeyouuseit,bestyieldsareobtainedatapHof8.5orgreater.The0.02micronAnotopsyringefiltersareavailablefromWhatman. 11.Ifdesired,repeatstep10toincreaseyield.Weobtain90%oftheDNAinthefirstelution. 12.QuantitateDNAsolution. 13.VerifysizeandintactconditionofDNAonminigel. 14.StoreelutedDNAat-20degreesCentigrade.