5/31/2016

Addgene:ProtocolHowtoRunanAgaroseGel

AgaroseGelElectrophoresis
BackgroundInformation

Youmayalsolike...

GelelectrophoresisisthestandardlabprocedureforseparatingDNAbysize(e.g.lengthinbasepairs)for
visualizationandpurification.ElectrophoresisusesanelectricalfieldtomovethenegativelychargedDNAtoward
apositiveelectrodethroughanagarosegelmatrix.ThegelmatrixallowsshorterDNAfragmentstomigratemore
quicklythanlargerones.Thus,youcanaccuratelydeterminethelengthofaDNAsegmentbyrunningitonan
agarosegelalongsideaDNAladder(acollectionofDNAfragmentsofknownlengths).

RestrictionDigestof
PlasmidDNA

Protocol:GelElectrophoresis

DNALigation

PurifyingDNAfroman
AgaroseGel

PouringaStandard1%AgaroseGel:
1.Measureout1gofagarose.
Note:Agarosegelsarecommonlyusedinconcentrationsof0.7%to2%dependingonthesizeofbandsneededtobeseparatedsee
FAQsbelow.Simplyadjusttheamountofstartingagaroseto%g/100mLTAE(i.e.2g/100mLwillgiveyou2%).
2.Pouragarosepowderintomicrowavableflaskalongwith100mLof1xTAE.
Note:SeeTAERecipe.
Note:TBEcanbeusedinsteadofTAE,labsusuallyuse
oneortheother,butthereisverylittledifferencebetween
thetwo.
3.Microwavefor13min(untiltheagaroseiscompletely
dissolvedandthereisanicerollingboil).
Note:CautionHOT!Becarefulstirring,eruptiveboiling
canoccur.
Note:Itisagoodideatomicrowavefor3045sec,stop
andswirl,andthencontinuetowardsaboil.Keepaneye
onitastheinitialboilhasatendencytoboilover.Placing
saranwrapoverthetopoftheflaskcanhelpwiththis,but
isnotnecessaryifyoupaycloseattention.
4.Letagarosesolutioncooldownfor5min.
5.(Optional)Addethidiumbromide(EtBr)toafinalconcentrationofapproximately0.20.5g/mL(usuallyabout23loflabstocksolutionper
100mLgel).EtBrbindstotheDNAandallowsyoutovisualizetheDNAunderultraviolet(UV)light.
Note:CautionEtBrisaknownmutagen.Wearalabcoat,eyeprotectionandgloveswhenworkingwiththischemical.
Note:IfyouaddEtBrtoyourgel,youwillalsowanttoaddittotherunningbufferwhenyourunthegel.IfyoudonotaddEtBrtothegel
andrunningbuffer,youwillneedtosoakthegelinEtBrsolutionandthenrinseitinwaterbeforeyoucanimagethegel.
6.Pourtheagaroseintoageltraywiththewellcombinplace.
Note:Pourslowlytoavoidbubbleswhichwilldisruptthegel.Anybubblescanbepushedawayfromthewellcombortowardsthe
sides/edgesofthegelwithapipettetip.
7.Placenewlypouredgelat4Cfor1015minutesORletsitatroomtemperaturefor2030minutes,untilithascompletelysolidified.
Note:Ifyouareinahurrythegelcanalsobesetmorequicklyifyouplacethegeltrayat4Cearliersothatitisalreadycoldwhenthe
gelispouredintoit.

LoadingSamplesandRunninganAgaroseGel:
1.Addloadingbuffertoeachofyourdigestsamples.
Note:Loadingbufferservestwopurposes:1)itprovidesavisibledyethathelpswithgelloadingandwillalsoallowsyoutogaugehowfar
thegelhasrunwhileyouarerunningyourgeland2)itcontainsahigh%glycerol,soafteraddingityoursampleisheavierthanwater
andwillsettletothebottomofthegelwell,insteadofdiffusinginthebuffer.
2.Oncesolidified,placetheagarosegelintothegelbox(electrophoresisunit).
https://www.addgene.org/plasmidprotocols/gelelectrophoresis/

1/2

5/31/2016

Addgene:ProtocolHowtoRunanAgaroseGel

3.Fillgelboxwith1xTAE(orTBE)untilthegeliscovered.
Note:Remember,ifyouaddedEtBrtoyourgel,addsometothebufferaswell.EtBrispositively
chargedandwillruntheoppositedirectionfromtheDNA.SoifyourunthegelwithoutEtBrinthe
bufferyouwillreachapointwheretheDNAwillbeinthebottomportionofthegel,butallofthe
EtBrwillbeinthetopportionandyourbandswillbedifferentiallyintense.Ifthishappens,youcan
justsoakthegelinEtBrsolutionandrinsewithwatertoevenoutthestainingafterthegelhas
beenrun,justasyouwouldifyouhadnotaddedEtBrtothegelinthefirstplace.
4.Carefullyloadamolecularweightladderintothefirstlaneofthegel.
Note:Whenloadingthesampleinthewell,maintainpositivepressureonthesampletoprevent
bubblesorbufferfromenteringthetip.Placetheverytopofthetipofthepipetteintothebuffer
justabovethewell.Veryslowlyandsteadily,pushthesampleoutandwatchasthesamplefills
thewell.Afterallofthesampleisunloaded,pushthepipettortothesecondstopandcarefully
raisingthepipettestraightoutofthebuffer.
5.Carefullyloadyoursamplesintotheadditionalwellsofthegel.
6.Runthegelat80150Vuntilthedyelineisapproximately7580%ofthewaydownthegel.
Note:Blackisnegative,redispositive.(TheDNAisnegativelychargedandwillruntowardsthe
positiveelectrode.)AlwaysRuntoRed.
Note:Atypicalruntimeisabout11.5hours,dependingonthegelconcentrationandvoltage.
7.TurnOFFpower,disconnecttheelectrodesfromthepowersource,andthencarefullyremovethegelfromthegelbox.
8.(Optional)IfyoudidnotaddEtBrtothegelandbuffer,placethegelintoacontainerfilledwith100mLofTAErunningbufferand5LofEtBr,
placeonarockerfor2030minutes,replaceEtBrsolutionwithwateranddestainfor5minutes.
9.UsinganydevicethathasUVlight,visualizeyourDNAfragments.
Note:WhenusingUVlight,protectyourskinbywearingsafetygogglesorafaceshield,glovesandalabcoat.
Note:IfyouwillbepurifyingtheDNAforlateruse,uselongwavelengthUVandexposeforasshortatimeaspossibletominimize
damagetotheDNA.
Note:ThefragmentsofDNAareusuallyreferredtoasbandsduetotheirappearanceonthegel.

AnalyzingYourGel:
UsingtheDNAladderinthefirstlaneasaguide(themanufacturer'sinstructionwilltellyouthesizeofeachband),youcaninterpretthebandsthat
yougetinyoursamplelanestodetermineiftheresultingDNAbandsthatyouseeareasexpectedornot.Formoredetailsondoingdiagnostic
digestsandhowtointerpretthempleaseseetheDiagnosticDigestpage.

PurifyingDNAfromYourGel:
Ifyouareconductingcertainprocedures,suchasmolecularcloning,youwillneedtopurifytheDNAawayfromtheagarosegel.Forinstructionson
howtodothis,visittheGelPurificationpage.

TipsandFAQ
Howdoyougetbetterresolutionofbands?
Afewsimplewaystoincreasetheresolution(crispness)ofyourDNAbandsinclude:a)runningthegelatalowervoltageforalongerperiodof
timeb)usingawidergelcomborc)loadinglessDNAintowell.
Howdoyougetbetterseparationofbands?
Ifyouhavesimilarlysizedbandsthatarerunningtooclosetogetheryoucanadjusttheagarosepercentageofthegeltogetbetterseparation.
Ahigherpercentageagarosegelwillhelpresolvesmallerbandsfromeachother,andalowerpercentagegelwillhelpseparatelargerbands.
10%Rule:
Foreachsampleyouwanttoloadonagel,make10%morevolumethanneededbecauseseveralmicroliterscanbelostinpipetting.For
example,ifyouwanttoload1.0gin10L,make1.1gin11L.
ReferencePage|Top|Index

https://www.addgene.org/plasmidprotocols/gelelectrophoresis/

2/2