MicroinjectionDNAPurification

WehavefoundtheNucleoSpin^TMExtractKit(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.

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