AGAROSE GEL ELECTROPHORESIS of DNA-豆柴文库

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AGAROSEGELELECTROPHORESISOFDNA1.ThephosphatemoleculesthatmakeupthebackboneofDNAmoleculeshaveahighnegativecharge.
2.TheDNAmoleculesarepulledtothepositiveendbythecurrent,buttheyencounterresistancefromthisagarosemesh.TheDNAmoleculesarepulledtothepositiveendbythecurrent,buttheyencounterresistancefromthisagarosemesh.
3.Thesmallermoleculesareabletonavigatethemeshfasterthanthelargerone,sotheymakeitfurtherdownthegelthanthelargermolecules.ThisishowagaroseelectrophoresisseparatesdifferentDNAmoleculesaccordingtotheirsize.DNA-(agiantanion)
cathode-(highnegativecharge)anode+Second:TheDNAmolecularisstainedwithethidium
bromidesoyoucanvisualizeaDNAbandsAgarosegelelectrophoresisseparatesDNAfragmentsaccordingtotheirsize.Typically,aDNAmoleculeisdigestedwithrestrictionenzymes,andtheagarosegelelectrophoresisisusedasadiagnostictooltovisualizethefragments.AnelectriccurrentisusedtomovetheDNAmoleculesacrossanagarosegel,whichisapolysaccharidematrixthatfunctionsasasortofsievetohelp“catch”themoleculesastheyaretransportedbytheelectriccurrent.Thistechniquehaslotsofapplications.ElectrophoresisProcedure（processorstep）PouringaGelThirdstep-Loadingsample：Fourthstep-ElectrophoresisFifthstepResult1.Priortogelcasting,driedagaroseisdissolvedinbufferbyheatingandthewarmgelsolutionthenispouredintoamold,whichisfittedwithawell-formingcomb.
2.Thepercentageofagaroseinthegelvaried.Although0.7%agarosegelstypicallyareused,incaseswheretheaccuratesizefractionationofDNAmoleculessmallerthan1kbisrequired,a1,1.5,or2%agarosegelisprepared,dependingontheexpectedsize(s)ofthefragment(s).
3.Agarosegelsaresubmergedinelectrophoresisbufferinahorizontalelectrophoresisapparatus.TheDNAsamplesaremixedwithgeltrackingdyeandloadedintothesamplewells.
4.Electrophoresisusuallyisat150-200mAfor0.5-1houratroomtemperature,dependingonthedesiredseparation.