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1、GelElectrophoresisofDNAWhatisGelElectrophoresis?Electro=flowofelectricity,phoresis,fromtheGreek=tocarryacrossAgelisacolloid,asuspensionoftinyparticlesinamedium,occurringinasolidform,likegelatinGelelectrophoresisreferstotheseparationofchargedparticleslocatedinagelwhenanelectr
2、iccurrentisappliedChargedparticlescanincludeDNA,aminoacids,peptides,etcWhydogelelectrophoresis?WhenDNAiscutbyrestrictionenzymes,theresultisamixofpiecesofDNAofdifferentlengthsItisusefultobeabletoseparatethepieces-I.e.forrecoveringparticularpiecesofDNA,forforensicworkorforsequ
3、encingWhatisneeded?Agarose-apolysaccharidemadefromseaweed.Agaroseisdissolvedinbufferandheated,thencoolstoagelatinoussolidwithanetworkofcrosslinkedmoleculesSomegelsaremadewithacrylamideifsharperbandsarerequiredBuffer-inthiscaseTBEThebufferprovidesionsinsolutiontoensureelectri
4、calconductivity.Notonlyistheagarosedissolvedinbuffer,butthegelslabissubmerged(submarinegel)inbufferafterhardeningAlsoneededareapowersupplyandagelchamberGelchamberscomeinavarietyofmodels,fromcommercialthroughhome-made,andavarietyofsizesHowdoesitwork?DNAisanorganicacid,andisne
5、gativelycharged(remember,DNAforNegative)WhentheDNAisexposedtoanelectricalfield,theparticlesmigratetowardthepositiveelectrodeSmallerpiecesofDNAcantravelfurtherinagiventimethanlargerpiecesAgelbeingrunAgaroseblockPositiveelectrodeDNAloadedinwellsintheagaroseBlackbackgroundTomak
6、eloadingwellseasierCombBufferStepsinrunningagelDNAispreparedbydigestionwithrestrictionenzymesAgaroseismadetoanappropriatethickness(thehigherthe%agarose,theslowerthebigfragmentsrun)and‘melted’inthemicrowaveThegelchamberissetup,the‘comb’isinsertedTheagarosemayhaveaDNA‘dye’adde
7、d(oritmaybestainedlater).TheagaroseispouredontothegelblockandcooledThecombisremoved,leavinglittle‘wells’andbufferispouredoverthegeltocoveritcompletelyTheDNAsamplesaremixedwithadenseloadingdyesotheysinkintotheirwellsandcanbeseenTheDNAsamplesareputinthewellswithamicropipette.M
8、icropipetteshavedisposabletipsandcanaccuratelymeasure1/1,000,000ofalitreNex
