琼脂糖凝胶电泳课件

琼脂糖凝胶电泳

Agarosegelelectrophoresis

一.电泳的概念带电质点在电场中向带有异相电荷的电极移动，这种现象称为电泳。区带电泳是在半固相或胶状介质上加一个点或一薄层样品溶液，然后加电场，分子在支持介质上或支持介质中迁移。支持介质的作用主要是为了防止机械干扰和由于温度变化以及大分子溶液的高密度而产生的对流。根据支持介质的材料:

琼脂糖电泳凝胶电泳

聚丙烯酰胺电泳二.实验目的1、掌握agarosegelelectrophoresis的原理

及操作方法.（一）琼脂糖凝胶电泳的原理琼脂糖是由琼脂分离制备的链状多糖。其结构单元是D-半乳糖和3.6-脱水-L-半乳糖。许多琼脂糖链依氢键及其它力的作用使其互相盘绕形成绳状琼脂糖束，构成大网孔型凝胶。该凝胶适合于免疫复合物、核酸与核蛋白的分离、鉴定及纯化。琼脂糖凝胶电泳分离核酸的基本原理：在一定浓度的琼脂糖凝胶介质中，DNA分子的电泳迁移率与其分子量的常用对数成反比；由于糖-磷脂骨架在结构上的重复性，相同数量的双链DNA几乎带有等量的静电荷，因此能以同样的速度迁移。分子构型也对迁移率有影响，如共价闭环DNA＞直线DNA＞开环双链DNA。（二）琼脂糖凝胶电泳的应用质粒DNA限制性酶切图谱的分析；DNA限制性酶切图谱的分析；PCR产物的分析；纯化特定的DNA片段；（三）影响电泳泳动率的四大因素1.样品的物理性状：分子大小、电荷多少、颗粒形状和空间构象双链DNA分子迁移的速率与其分子量常用对数近似成反比PlasmidDNAI型（闭环CC），超螺旋

II型（单链开环OC），松弛

III型（线性L）。

I型

III型

II型2.支持物介质（agarose,PAGE)：浓度越低，相同核酸分子迁移越快；分离小于0.5kb的DNA片段所需胶浓度是1.2-1.5%,分离大于10kb的DNA分子所需胶浓度为0.3-0.7%,DNA片段大小间于两者之间则所需胶浓度为0.8-1.0%。3.电场强度：低电压时DNA片段迁移率与所用的电压成正比。4.缓冲液离子强度：

TAE,TBE,TPE在没有离子存在时(如误用蒸馏水配制凝胶),电导率最小,DNA几乎不移动,在高离子强度的缓冲液中(如误加10×电泳缓冲液),则电导很高并明显产热,严重时会引起凝胶熔化或DNA变性。TAE、TPE及TBE电泳缓冲液比较都是常用电泳缓冲液。三者相比：1）TAE的缓冲容量最低，如长时间电泳会被消耗，此时凝胶的阳极一侧将发生酸性化；2）TBE和TPE比TAE花费稍贵，但有高得多的缓冲容量；3）双链线状DNA片段在TAE中比在TBE或TPE中迁移快10%；4）对于高分子质量的DNA，TAE的分辨率略高于TBE或TPE，对于低分子质量的DNA，TAE要差些。超螺旋DNA在TAE中的电泳分辨率要好于TBE。核酸电泳的指示剂溴酚蓝（bromophenolblue)二甲苯青蓝FF（xylenecyanol)6×凝胶载样缓冲液类型6×缓冲液贮存温度I0.25%溴酚蓝4℃0.25%二甲苯氰FF40%(m/V)蔗糖水溶液II0.25%溴酚蓝室温0.25%二甲苯氰FF15%Ficoll(Type400)水溶液III0.25%溴酚蓝4℃0.25%二甲苯氰FF30%甘油水溶液IV0.25%溴酚蓝4℃40%(m/V)蔗糖水溶液上样缓冲液(Loadingbuffer）：作用：1）增加样品密度保证DNA沉入加样孔内:10%-15%蔗糖或5%~10%甘油。2）使样品带有颜色便于简化上样过程。3）其中的染料在电场中以可以预测的泳动速率向阳极迁移:0.25%溴酚蓝或其他指示染料。（四）琼脂糖凝胶电泳装置琼脂糖凝胶电泳的观察在琼脂糖凝胶中加入溴化乙锭（ethidiumbromide,EB）或Goldview。在紫外光下就可观察到明显DNA的荧光谱带。Goldview:在紫外透射光下双链DNA呈现绿色荧光，而单链DNA呈红色荧光（五）琼脂糖凝胶电泳的基本步骤

和注意事项1、制胶2、上样3、观察4、拍照EB—CarcinogenUltraViolet—hurtyoureyes实验仪器电泳仪，电泳槽，电子天平，移液器，电磁炉，紫外透射检测仪等。试剂琼脂糖，1XTAE电泳缓冲液，goldview

6X载样缓冲液Moreagarosemeansafirmergel,andamoredifficultmatrixfortheDNAtomovethrough.Gelsareoftenmixedat0.8-1.0%agarose.Thisconcentrationwillseparateawiderangeoffragmentsizes,rangingfromaround500basepairs(bp)toaround20,000bp.

PerhapsyouhaveseenthetermsTBEorTAE.Thesearenamesoftwocommonlyusedbuffersinelectrophoresis.The"T"standsforTris,achemicalwhichhelpsmaintainaconsistentpHofthesolution.The"E"standsforEDTA,whichitselfisanotheranacronym.EDTAchelates(gobblesup)divalentcationslikemagnesium.Thisisimportantbecausemostnucleasesrequiredivalentcationsforactivity.Finally,the"B"or"A"standforBoricacidorAceticacid,whichprovidetheproperionconcentrationforthebuffer.Thesolventusedtodissolvetheagarose.Ratherthanjustusewater,weusebufferedsolutionswhichallowtheDNAtorunsmoothlythroughthegel.ThesesolutionsoptimizethepHandionconcentrationofthegelandwillalsobathethegelasitissubjectedtotheelectriccurrentwhichactuallymovestheDNAthroughthegel.Aftertheagarosehasbeenweighedandthebuffermeasured,thetwoaremixed.itmustbeboiled.Thefastestwaytodothisisbyputtingtheflaskinthemicrowave.

InordertomakeourDNAmigratethroughthegel,weneedtomakesuretheagarosepolymerizeswithsmall"wells"initinwhichtoputtheDNA.Thisisthejobofthecomb,Thecombisplacedintoslotsinthetray,withthe"teeth"down,whentheagarosecoolsandhardens,thecombisgentlyliftedupoutofthegel,leavingthespacesoccupiedbytheteeth.Thisgivesusseveralplacestoloaddifferentsamples.Thecastingapparatusconsistsof3parts--thetray,thesupport,andthecomb.Thetrayistheactualmoldwhichprovidesashapeforthegelasitpolymerizes.

Youcanseethemoltenagaroseadvancingacrossthesurfaceofthesupportasitispoured.Atroomtemperature,thisgelwillhavepolymerizedenoughtousewithin10-15minutes.Thecastingtraycouldhold2gelsatonce,althoughonlyoneisbeingpouredatthistime.Hereyouseethehotagarosewaspouredintothecastingtray.Theglasssupportispreventingtheagarosefromleakingoutthroughtheholesinthebottomofthetray.Inbothofthesephotos,agelwhichhascompletelypolymerizedisnowreadytouse.Noticethespaces(called"wells")leftbytheremovalofthecomb.Thiscanbeseenbetterinthesecondphoto.Bothgelsarerestingontheglasssupport.Thehighertheagarosepercentage,thebluerthegel.The"runningtank"wasfilledwithbuffer.IfTBEwasusedtomakethegel,itshouldalsobeusedastherunningbuffer.Thegelwillbecompletelysubmergedasitisrun.Noticethetwometalpinsmarkedbythearrows.Thesearetheconnectionplugsforthepowersupplycables.ItisthiscurrentthatmovestheDNAthroughthegel.IsDNApositivelyornegativelycharged?Towardwhichelectrodedoyouthinkitwillmigrate?

First,Glycerolisindeedused,becauseithasadensitygreaterthanwater.Next,tomonitorourDNAasitmigratesacrossthegel.wecanuseddyes,thatareoftwodifferentsizesandcolors.Wecanthereforeassumethatoursampleissomewhereinbetweenthedyes.NowtheDNAsampleismixedwithLoadingBuffer,Oursampleisnowreadytoload.ThisisaviewfromthetopoftherunningtankastheagarosegelisloadedwiththeDNAsamples.Oncethegelhasbeenloaded,theelectricalleadsontherunningtankareconnectedtoapowersupplyliketheoneshowninthephoto.Thepowersupplyhas3needlegaugesonit,showingwhatthevoltage,current,andpowerlevelsare.Powersupplieslikethiscanberuninconstantmodeforanyofthe3variables,andthedialsbelowtheknobsareusedtosettheconstantlevel.Afterwehaveremovedourgelfromtherunningtank,weneedtouseultravioletlighttovisualizetheDNAandverifythatourRestrictionDigestwassuccessful.EtBrisanIntercalatingAgent,meaningitwedgesitselfintothegroovesofDNAandstaysthere.Morebasepairsmeanmoregooves,whichinturnmeansmoreEtBrcaninsertitself.EtBralsohasthepropertyoffluorescingunderUVlight.wecan"see"theDNAbyactuallydetectingthefluorescenceoftheEtBr.Inthisparticularphotograph,Lanes1-3containourDNAsamples,perhapsfromthesamestockofDNAdigestedwith3differentRestrictionEnzymes,Whenwewanttocalculatethesizeofthesebands.Howcanwedothis?Weusetheinformationinlanes4and5,andalittlemath.Lanes4and5arecalledStandardLanes,orMolecularWeightMarkers.Lane5isDNAfromtheLambdaDNA,digestedwithHin

dIII.TovisualizetheDNAandverif