AGAROSE GEL ELECTROPHORESIS of DNA.ppt

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1、AGAROSEGELELECTROPHORESISOFDNA1.ThephosphatemoleculesthatmakeupthebackboneofDNAmoleculeshaveahighnegativecharge.2.TheDNAmoleculesarepulledtothepositiveendbythecurrent,buttheyencounterresistancefromthisagarosemesh.TheDNAmoleculesarepulledtothepositiveen

2、dbythecurrent,buttheyencounterresistancefromthisagarosemesh.3.Thesmallermoleculesareabletonavigatethemeshfasterthanthelargerone,sotheymakeitfurtherdownthegelthanthelargermolecules.ThisishowagaroseelectrophoresisseparatesdifferentDNAmoleculesaccordingto

3、theirsize.PRINCIPLEFirst:AgarosegelelectrophoresisSeparatesDNAfragmentsaccordingtotheirsizebymoleculessieve.DNA-(agiantanion)cathode-(highnegativecharge)anode+ElectricalfieldStructure:across-linkedmatrix(Namelyathree-dimensionalmesh)Function:moleculesi

4、eveeffectResistanceElectricalforceAgarosegelSecond:TheDNAmolecularisstainedwithethidiumbromidesoyoucanvisualizeaDNAbandsEthidiumbromideisanintercalcatingdye,whichmeansitinsertsitselfbetweenthebasesthatarestackedinthecenteroftheDNAhelix.Oneethidiumbromi

5、demoleculebindstoonebase.Aseachdyemoleculebindstothebasesthehelixisunwoundtoaccommodatethestrainfromthedye.DNAhelixEthidiumbromideisanintercalcatingdyeOneethidiumbromidemoleculebindstoonebase100ml1%Agaroseadd100ul10%EthidiumbromideorafterElectrophoresi

6、sAgarosegeldipin100ml1×TAEbuffer(contain100ul10%EB)10min.AgarosegelelectrophoresisseparatesDNAfragmentsaccordingtotheirsize.Typically,aDNAmoleculeisdigestedwithrestrictionenzymes,andtheagarosegelelectrophoresisisusedasadiagnostictooltovisualizethefragm

7、ents.AnelectriccurrentisusedtomovetheDNAmoleculesacrossanagarosegel,whichisapolysaccharidematrixthatfunctionsasasortofsievetohelp“catch”themoleculesastheyaretransportedbytheelectriccurrent.THIRD-APPLICATION:SEPARATINGTHEDNAFRAGMENTSOFENDONUCLEASESANDPC

8、RREACTIONThistechniquehaslotsofapplications.GenerallyspeakingyoucananalyzeDNAfragmentsthatresultfromanenzymedigestionofalargerpieceofDNAtovisualizethefragmentsanddeterminethesizesofthefragments.Inadditiontoitsusefulnessinresearchtechniq