瓊脂糖凝膠電泳

Agarosegelelectrophoresis

一.電泳的概念帶電質(zhì)點在電場中向帶有異相電荷的電極移動，這種現(xiàn)象稱為電泳。區(qū)帶電泳是在半固相或膠狀介質(zhì)上加一個點或一薄層樣品溶液，然后加電場，分子在支持介質(zhì)上或支持介質(zhì)中遷移。支持介質(zhì)的作用主要是為了防止機(jī)械干擾和由于溫度變化以及大分子溶液的高密度而產(chǎn)生的對流。根據(jù)支持介質(zhì)的材料:

瓊脂糖電泳凝膠電泳

聚丙烯酰胺電泳二.實驗?zāi)康?、掌握agarosegelelectrophoresis的原理

及操作方法.（一）瓊脂糖凝膠電泳的原理瓊脂糖是由瓊脂分離制備的鏈狀多糖。其結(jié)構(gòu)單元是D-半乳糖和3.6-脫水-L-半乳糖。許多瓊脂糖鏈依氫鍵及其它力的作用使其互相盤繞形成繩狀瓊脂糖束，構(gòu)成大網(wǎng)孔型凝膠。該凝膠適合于免疫復(fù)合物、核酸與核蛋白的分離、鑒定及純化。瓊脂糖凝膠電泳分離核酸的基本原理：在一定濃度的瓊脂糖凝膠介質(zhì)中，DNA分子的電泳遷移率與其分子量的常用對數(shù)成反比；由于糖-磷脂骨架在結(jié)構(gòu)上的重復(fù)性，相同數(shù)量的雙鏈DNA幾乎帶有等量的靜電荷，因此能以同樣的速度遷移。分子構(gòu)型也對遷移率有影響，如共價閉環(huán)DNA＞直線DNA＞開環(huán)雙鏈DNA。（二）瓊脂糖凝膠電泳的應(yīng)用質(zhì)粒DNA限制性酶切圖譜的分析；DNA限制性酶切圖譜的分析；PCR產(chǎn)物的分析；純化特定的DNA片段；（三）影響電泳泳動率的四大因素1.樣品的物理性狀：分子大小、電荷多少、顆粒形狀和空間構(gòu)象雙鏈DNA分子遷移的速率與其分子量常用對數(shù)近似成反比PlasmidDNAI型（閉環(huán)CC），超螺旋

II型（單鏈開環(huán)OC），松弛

III型（線性L）。

I型

III型

II型2.支持物介質(zhì)（agarose,PAGE)：濃度越低，相同核酸分子遷移越快；分離小于0.5kb的DNA片段所需膠濃度是1.2-1.5%,分離大于10kb的DNA分子所需膠濃度為0.3-0.7%,DNA片段大小間于兩者之間則所需膠濃度為0.8-1.0%。3.電場強(qiáng)度：低電壓時DNA片段遷移率與所用的電壓成正比。4.緩沖液離子強(qiáng)度：

TAE,TBE,TPE在沒有離子存在時(如誤用蒸餾水配制凝膠),電導(dǎo)率最小,DNA幾乎不移動,在高離子強(qiáng)度的緩沖液中(如誤加10×電泳緩沖液),則電導(dǎo)很高并明顯產(chǎn)熱,嚴(yán)重時會引起凝膠熔化或DNA變性。TAE、TPE及TBE電泳緩沖液比較都是常用電泳緩沖液。三者相比：1）TAE的緩沖容量最低，如長時間電泳會被消耗，此時凝膠的陽極一側(cè)將發(fā)生酸性化；2）TBE和TPE比TAE花費稍貴，但有高得多的緩沖容量；3）雙鏈線狀DNA片段在TAE中比在TBE或TPE中遷移快10%；4）對于高分子質(zhì)量的DNA，TAE的分辨率略高于TBE或TPE，對于低分子質(zhì)量的DNA，TAE要差些。超螺旋DNA在TAE中的電泳分辨率要好于TBE。核酸電泳的指示劑溴酚藍(lán)（bromophenolblue)二甲苯青藍(lán)FF（xylenecyanol)6×凝膠載樣緩沖液類型6×緩沖液貯存溫度I0.25%溴酚藍(lán)4℃0.25%二甲苯氰FF40%(m/V)蔗糖水溶液II0.25%溴酚藍(lán)室溫0.25%二甲苯氰FF15%Ficoll(Type400)水溶液III0.25%溴酚藍(lán)4℃0.25%二甲苯氰FF30%甘油水溶液IV0.25%溴酚藍(lán)4℃40%(m/V)蔗糖水溶液上樣緩沖液(Loadingbuffer）：作用：1）增加樣品密度保證DNA沉入加樣孔內(nèi):10%-15%蔗糖或5%~10%甘油。2）使樣品帶有顏色便于簡化上樣過程。3）其中的染料在電場中以可以預(yù)測的泳動速率向陽極遷移:0.25%溴酚藍(lán)或其他指示染料。（四）瓊脂糖凝膠電泳裝置瓊脂糖凝膠電泳的觀察在瓊脂糖凝膠中加入溴化乙錠（ethidiumbromide,EB）或Goldview。在紫外光下就可觀察到明顯DNA的熒光譜帶。Goldview:在紫外透射光下雙鏈DNA呈現(xiàn)綠色熒光，而單鏈DNA呈紅色熒光（五）瓊脂糖凝膠電泳的基本步驟

和注意事項1、制膠2、上樣3、觀察4、拍照EB—CarcinogenUltraViolet—hurtyoureyes實驗儀器電泳儀，電泳槽，電子天平，移液器，電磁爐，紫外透射檢測儀等。試劑瓊脂糖，1XTAE電泳緩沖液，goldview

6X載樣緩沖液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