琼脂糖凝胶电泳

琼脂糖凝胶电泳凝胶制备DNAMarker加样与电泳观察与记录本卷须知AGAROSE强度600,1000~1200,1500熔点65◦C,86~88◦C凝固26~30◦C,36~38◦C电渗(Electroendosmosis)EEO/mr用途：100bp~2kb

AmpliAg.1kb~10kbMolecularBio.Ag.105~106bpPulsedfieldcertifiedAg.>106bpChromosomalDNAAg.DNAMarkerLamdaDNA/Re1kb,100bp,50,10bpladder(Molecularbio,amplification)Pulseele.MarkerChromosomalMarkerTheappropriatemarkermatchingDNAbandsMeltingtime

0.7~1.5%5min(Microwaveoven)无折光颗粒，均匀冷却与凝胶

充足的时间，20min,氢键,网状结构EB0.1~0.5ug/uL(positivecharge)chamber,soak1~2mmLoadingbufferBubblewellCellTray

TAEbufferprovidesfasterelectrophoreticmigrationoflinearDNAandbetterresolutionofsupercoiledDNA

TBEbuffershaveastrongerbufferingcapacityforlongerorhighervoltageelectrophoresisruns.EthidiumBromideStainingProcedure

1.Placethegelintotheappropriatevolumeof0.5µg/mlethidiumbromide(EtBr)stainfor15–30minutes.Useenoughstainingsolutiontocovertheentiregel.Caution:Ethidiumbromideisasuspectedcarcinogenandshouldbehandledwithextremecare.Alwaysweargloves,eyeglasses,andalaboratorycoat.DisposeofusedEtBrsolutionsandgelsappropriately2.Destainthegelfor10–30minutesindH2Ousingthesamevolumeusedforstaining.Note:EthidiumBromidecanberemovedfromtheDNAwithextendeddestaining.Thiswillcauselowersensitivityofdetection.However,insufficientdestainingwillcreatehigherbackgroundfluorescence.3.RinsethegelbrieflywithdH2Otoremoveanyresidualstainingsolution.4.PlacethegelonaUVtransilluminatorfornucleicacidvisualizationandanalysis.DNAEthidiumBromidecomplexesmaybeilluminatedwithUVlightof254,302,or366nm.Sensitivitydecreaseswithilluminationathigherwavelengths.However,nickingofDNAwillincreasebelow302nm.Table2.5givesthepercentageoftransmittanceofUVlightthrough1/4〞(.64cm)UV-transparentplastic.11DNAtoomchImpurityGelproblem,[C]?Strength?Broken?VoltageRunningBufferNoDNA,degradationExtractionmethodSaltsOthers(DNA-RE/PTcomplex)?TroubleshootingRunningBufferTAE,1XTBE,0.5XTPE?VoltageandCurrent

5V/cm,lower,betterS