Beruflich Dokumente
Kultur Dokumente
GELELECTROPHORESISOFDYES
PartnershipforBiotechnologyandGenomicsEducation
BarbaraSoots
LindaCurro
EducationCoordinator
AssistantEducationCoordinator
UniversityofCaliforniaDavis
UniversityofCaliforniaDavis
5307526552
5307526613
5307544410(fax)
5307544410(fax)
besoots@ucdavis.edu
llcurro@ucdavis.edu
Thisprogramismadepossiblethroughthegeneroussupportof:
GEL ELECTROPHORESIS
Dye Samples
Introduction
Inthisexperiment,studentswillbeusing
agarosegelelectrophoresistoseparateseveral
dyesaprecursortotheDNAseparationswe
willperforminlaterlabs.
Thislabwillhelpstudentsgainfamiliaritywith
equipmentandtechniquesessentialto
biotechnology.Theywilllearntocastandload
anagarosegelaswellascorrectlyutilizegel
electrophoresisequipment.Attachedreadings
andactivitieswillbetterillustratethisimportant
technique.
electrophoresisgelbox
1geltraywith6toothcomb
50mlbeakerorgraduatedcylinder*
disposableplasticpipette
250mlbeaker*
20lmicropipetwithtips
gloves
safetyglasses*
papertocoverlabbench*
CommonMaterials
powersupplies
60Cwaterbath
0.7%agarosesolution(enoughfor25
mlperteam)
plasticcarboywithTBEbuffer(1X)
geldisposalbags
distilledwater*
Note:Yourstudentsshouldhavecompletedthe
MicropipetTechnique:DyeSampleslabprior
torunningthisexperiment.
Objectives
Teacher
Information
1. Understandtheprinciplesbehindgel
electrophoresis.
2. Becomefamiliarwithpreparing,loading
andrunningagel.
3. Determinetheeffectofamolecule's
electricchargeandsizeonitsmovement
throughanagarosegel.
4. Determinethecomponentsofanunknown
dyemixture
*Materialsprovidedbyinstructor.
Materials
Figureonabout300mlpergroup3,600mlifyour
classhastwelvegroups.
M1V1=M2V2
(molarityofbuffer)(vol.buffer)=
(1Xbuffer)(totalvol.ofrxn.)
(10)(x)=(1)(3,600ml)
x=360ml
Use360mlof(10X)bufferandadddistilledwaterto
bringthefinalconcentrationto3,600ml.
ForEachLabGroup
Samplesofthefollowingdyes:
Bromophenolblue
OrangeG
PyroninY
SafraninO
XyleneCyanol
Unknownmixture
microtuberack
AdvancePreparation
1.
DilutetheconcentratedTBE(10X)solution
toafinalconcentrationof(1X)andstorein
aplasticcarboyforeasyaccessby
students.Afterthelab,thesolutionmaybe
pouredbackintothecarboyandreusedfor
theRestrictionDigestLaboratory.
2.
3.
Loosenthelidfromthebottleof0.7%
agarose.Microwaveinafoodsafeoven
untilthegelismeltedwatchcarefully!
Then,immersethebottleinadistilledhot
waterbathtokeepliquiduntilneededby
thestudents.Studentswillneedtohandle
thebottlessohavepotholdersavailable.
USEOFPOWERSUPPLIES:
Aliquot25lsamplesofthe6dyesin
microtubes.Twogroupsmayshareoneset
ofdyes.
TeacherNotes
USEOFMICROPIPETS:
SeenotesinMicropipetTechnique
laboratory
BUFFERSOLUTION:
Tris/Borate/EDTA(TBE)bufferiscommonly
usedinelectrophoresissystems.Thissalt
solutionbothconductstheelectriccurrent
andcontrolsthepHofthesolutionduring
separationofDNAfragments,orinthiscase,
dyemolecules.
DISTILLEDWATER:
Mineralsinregulartapwaterwillquickly
stainequipment.Pleasehaveyourstudents
rinseanddryboththegeltraysandgel
boxesindistilledwater.Becarefulnotto
dislodgethewiringatthebaseofthegelbox
duringthedryingprocess.
GELDISPOSAL
Thesegelsmaybedisposedofintheregular
trash.
Whentwogelboxesarerunningoffofone
powersupply,expectalotofcondensationon
theboxlids.
Thepowersupplyproducesavoltagethatis
highenoughtocausesevereelectricalshockif
handledimproperly.
DONOTplugpowersupplyintowall
receptacleuntilthesafetycoveris
positionedonthecellandallother
electricalconnectionsareproperly
made.
Thisunitusesa3wiregroundedplug.
Forsafetyreasons,itshouldNOTbe
usedwith2wirereceptaclewitha
conversionplug.
DONOToperateinadamporhumid
environmentanycondensed
moisturemayshortoutelectrical
components.Makesurethatall
electricalequipmentisdry.
Inspectallpowercords,patchcords,
bananajacksandplugsforanydefects,
suchascrackedanddriedout
insulationandlooseorwobblybanana
jacksorplugs.
DONOTcomeinpersonalcontactwith
orallowmetaloranyconductive
materialtocomeincontactwith
reservoirbufferortheelectrophoretic
cellwhilepowersupplyison.
Thepowersupplymaycontinueto
producesomevoltageevenwhenthe
powerhasbeenturnedoff.To
eliminateanyriskassociatedwiththis
event,followallrequiredstepsgivenin
theprocedure.Whendisconnecting
thegelboxbesurethattheleadsdo
nottoucheachother,comeincontact
withthebuffersolution,orotherwise
createanyhazardouselectrical
condition.
BiotechnologyintheClassroom2009
updated9/23/2009
AnswerstoStudentActivity
1.
gelbox
comb
+
geltray
2. Thedyeshavebothpositiveandnegativechargesandwillmigrateindifferentdirections.
3.
Bromophenol Blue
purple
Orange G
yellow
Pyronin Y
pink
Safranin O
red
Xylene Cyanol
aqua
4. BromophenolBlue,OrangeG,andXyleneCyanol.
5. OrangeG
6. BromophenolBlue,OrangeG,XyleneCyanol.
7. Gelelectrophoresis:atechniqueusedtoseparatechargedmolecularfragmentsinamatrix
suchasagarose,byapplyinganelectriccurrent.Fragmentscanbeseparatedbysize,because
smallfragmentsmigratequickerthanlargefragments.
BiotechnologyintheClassroom2009
updated9/23/2009
GEL ELECTROPHORESIS
Dye Samples
Laboratory
Background
Oneofthemostbasicandfrequentlyusedtoolsofthemolecularbiologistiselectrophoresis.Infact,
DNAfingerprintingisbasedonthisprocess.Electrophoresiscanbeusedtoseparatemoleculesof
differentsizesandelectricalcharges,mostoftenfragmentsofproteinorDNA.Inthislab,however,we
willbeusingcoloreddyes.Toperformgelelectrophoresisthreethingsareneededamediumin
whichtoseparatethemolecules,anelectricalcurrent,andabuffersolution.
Duringagaroseelectrophoresis,molecularfragments
migratethroughagelwhenexposedtoelectricity.Agarose
isaJellOlikesubstancederivedfromseaweed.Thegel
hasarelativelylargeporesize,makingitusefulforsize
separationofmolecules.Inthegelareseveralsmallwells,
madebytheteethofacombplacedinthegelbeforeit
set.Samplestobetestedareplacedinthewellswitha
micropipet.Therateofmovementofeachsample
dependsuponmoleculesizeandshape.Inthepresenceof Aspongemakesagoodcomparisontoagel.Largeandsmall
particleswouldpassthroughaspongeatdifferentrates
anelectricalcurrent,smallmoleculesmovequickly
accordingtoparticlesizeversusthesizeoftheholesinthe
throughtheporousgel.Largemoleculesfinditmore
sponge.
difficulttomove,sotheytravelmoreslowly.Thedirection
ofmovementdependsontheelectriccharge(+or)ofthe
molecules,andthedensityofthegelthroughwhichtheDNAmoves.SinceDNAmoleculesallhavethe
samecharge,theyallmoveinthesamedirectiontowardspositive.Today,however,youwillbe
separatingdifferentcolordyes;somehaveapositivechargeandothersanegativecharge.
Whenthegelisplacedinagelbox,abuffersolutionispouredoverthegel,sothatitfillsthewellsand
makescontactwiththeelectrodesateachendofthegelbox.Saltsdissolvedinthebuffersolution
conductelectricityacrossthegel.Thebufferalsostopsthegelfromdryingout.Priortoloadingthe
samplesinthewellsofthegel,themoleculestobeseparatedaremixedwithadensesugarsolution,
sothatwhenthemixtureisaddedtothewells,itsinkstothebottom,takingthetestmolecules(in
thiscase,dye)withit.Withoutthesugar,thetestmoleculeswouldsimplyfloatawayinthebuffer.
Afterthegelhasbeenexposedtotheelectricalcurrentinthisparticularexperiment,about20
minutesthedyescanbeseenonthegel.However,rememberthatwhenusinginvisibleDNAor
proteinfragments,thegelmustbestainedorspeciallytreatedtorevealthefamiliar"barcode"
patterns.
Attachedreadingsandactivitiesinthelabmanualwillhelptoclarifytheprocessofelectrophoresis.
BiotechnologyintheClassroom2009
updated9/23/2009
gel tray
agarose gel
negative electrode
positive electrode
cathode
anode
platinum wire
platform for gel tray
DiagramofGelBoxandTray
Objectives
1.
2.
3.
4.
Understandtheprinciplesbehindgelelectrophoresis.
Becomefamiliarwithpreparing,loadingandrunningagel.
Determinetheeffectofamolecule'selectricchargeandsizeonitsmovementthroughanagarosegel.
Determinethecomponentsofanunknowndyemixture
Materials
CommonMaterials
ForEachLabGroup
powersupplies
60Cwaterbath
0.7%agarosesolution(enoughfor25ml
perteam)
plasticcarboywithTBEbuffer(1X)
geldisposalbags
distilledwater*
Samplesofthefollowingdyes:
Bromophenolblue
OrangeG
PyroninY
SafraninO
XyleneCyanol
Unknownmixture
microtuberack
electrophoresisgelbox
1geltraywith6toothcomb
50mlbeakerorgraduatedcylinder*
disposableplasticpipette
250mlbeaker*
20lmicropipetwithtips
gloves
papertocoverlabbench*
safetyglasses*
BiotechnologyintheClassroom2009
updated9/23/2009
Materialsprovidedbyinstructor.
Precautions
FollowdirectionsinMicropipetTechnique
laboratoryregardingmicropipethandling.
Thepowersupplyproducesavoltagethat
ishighenoughtocausesevereelectrical
shockifhandledimproperly.Assurethat
youarethoroughlydrilledinthe
proceduresregardingtheuseofthisunit,
makingelectricalconnections,andthat
youaredirectlysupervisedbythe
teacher.
DONOTplugpowersupplyintowall
receptacleuntilthesafetycoveris
positionedonthecellandallother
electricalconnectionsareproperlymade.
Makesurethatallelectricalequipment
isdry.
DONOTcomeinpersonalcontactwithor
allowmetaloranyconductivematerialto
comeincontactwithreservoirbufferor
theelectrophoreticcellwhilepower
supplyison.
Thepowersupplymaycontinueto
producesomevoltageevenwhenthe
powerhasbeenturnedoff.When
disconnectingthegelboxbesurethatthe
leadsdonottoucheachother,comein
contactwiththebuffersolution,or
otherwisecreateanyhazardouselectrical
condition.
BiotechnologyintheClassroom2009
updated9/23/2009
Procedure
1.
2.
3.
4.
5.
6.
7.
8.
Gatherallnecessarymaterials.Putonglovesandsafetyglasses.Youwillbesharingdye
samplesandapowersupplywithanotherlabgroup.
Makesurethesidesareuponyourgeltray.Ifnecessary,usetapeto
sealtheends.Placetheplasticcombinthemiddleofthetray.Cover
yourlabspacewithpaperpriortopouringthegel.
Gothehotwaterbathandfilla50mlbeakerwith2530mlofmelted
agarose.
Usingadisposableplasticpipette,runathinlineofagarosearoundthe
inneredgeofyourgeltray.
Pourtheremainingagaroseintothegeltray.Liquidshouldbeapproximately1/3oftheway
uptheplasticteethofthegelcomb.Removeanybubblesinthesolutionwithadisposable
pipette.
Letthegelhardenonyourdeskforatleast15minutes.
Oncesolidified,makesurethatthegatesaredownorthatthetapeisremovedfromtheends
ofthetray.Carefullyremovethecombbypullingstraightoutofthesolidifiedgel.Placeyour
geltrayonthecenterislandofthegelbox.
Obtainapproximately250mlofTBEbufferfromtheplasticcarboy.Pourthebufferintothe
sidecompartmentsofthegelboxtocompletelyfilltheboxandtocoverthetopgelsurface
withabout2mmofsolution.Donotpourbufferdirectlyontothewellareaofthegel!After
fillinggelbox,pouranyremainingTBEbufferbackintothecarboy.
STOPPOINT:Ifthereisnotenoughtimetorunthegelinthislabperiod,gelboxescanbe
closedandleftuntilthenextclassmeeting.
9.
Onthegel,load10lofeachdyeintoacorrespondinglane.Keeptrackofwhichdyegoesinto
whichlaneontheaccompanyingactivitysheet.Useanewtipforeachdyeandbecarefulnot
topuncturethebottomofthewell.Yourteacherwilldemonstrate.
Lane#1
Lane#2
Lane#3
BromophenolBlue
OrangeG
PyroninY
Lane#4SafraninO
Lane#5XyleneCyanol
Lane#6Unknowndyemixture
Inserttipofmicropipetjustoverthetopofthewell andejectdye.Thedyesareloadedwitha
sucrosesolutionandwillsink.Ifyoupushthetipdowntoofar,youmaypuncturethewell.
BiotechnologyintheClassroom2009
updated9/23/2009
10.
Cleanupanyspilledbufferoranyotherliquidsurroundingthegelboxthoroughly.
11.
Makesurethatthepowersupplyisunpluggedandswitchedoffbeforeproceeding.
12.
Carefullyplacethelidonthegelbox(firstmakingsureitiscleananddry).
13.
Connectthered(positive)patchcordtotheredterminalonthepowersupply.Similarly,
connecttheblackcordtotheblackterminal.Eachpowersupplywillruntwogelboxes.Notice
whatchannelyouhavepluggedyourboxinto.
14.
Waituntilthegroupthatissharingyourpowersupplyhascompletedsteps112before
proceeding.
15.
IMPORTANT!Haveyourteacherlookoverallofyourconnectionsbeforeplugginginthe
powersupply.
16.
Pluginthepowersupplyandturnonthemachineon.Therunlightwillilluminate,signifying
thatpowerisrunningtothecell.
17.
Turnthedisplayselectswitchtotheappropriatechannelforyourgroup.Turnthevoltage
indicatorknobto100V.Makesurebothgroupssettheirvoltage.Observethebubblesthat
formalongtheplatinumelectrodes.
18.
Letyourgelrunundisturbedfor2030minutes.Keepcheckingyourgeltoassurethatthe
dyesdonotrunofftheendofthegelintothebuffercompartment.Duringthistimecomplete
theattachedactivitysheet.
19.
Whenthedyesreachapproximately2cmfromtheendsofthegel,turnoffthepowersupply.
Thendisconnectthepatchcordsfromthepowersupplyandunplugtheunit.
20.
Makesureyourglovesareon.Removeyourgeltrayfromthegelboxcarefully.Tiptrayover
gelboxtodiscardexcessbuffersolution.Recordyourobservationsontheactivitysheet.
21.
Followyourteachersinstructionsforcleanup.Micropipettipsandgelsmaybediscardedin
trash.Gelboxesandtraysmustberinsedindistilledwaterandairdried.Donotusepaper
toweltodryinsidetheboxes!Pleasebecarefulwiththeequipment.
22.
Washhandsthoroughlyandcleanlabstationbeforeleavingthelab.
BiotechnologyintheClassroom2009
updated9/23/2009
GEL ELECTROPHORESIS
Dye Samples
Student
Activity
Name:________________________________________________________________________
ThesidesoftheDNAladderaremadeupofalternatingsugarandphosphategroups.Thesephosphate
groups(PO4)giveDNAanoverallnegativecharge.
1. IfyouwerepouringagelforDNAagarosegelelectrophoresis,wherewouldyouplacethecomb?
Drawapicturebelow.
gelbox
+
geltray
2. Whyisthecombplacedinthecenterforthisdyeelectrophoresisexperiment?
3. Thediagrambelowrepresentsyourgel.Usingcoloredpencilsormarkers,drawintheresultsof
yourdyeseparation.Besuretolabeleachlanewiththenameofthedyethatwas
electrophoresed.
BiotechnologyintheClassroom2009
updated9/23/2009
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4. Whatdyeswereintheunknownmixture?
5. Whichnegativelychargeddyeseemstobethesmallestmolecule?
6. Listthedyesthathaveanegativecharge.
7. Inyourownwords,explainhowagarosegelelectrophoresisworks.
BiotechnologyintheClassroom2009
updated9/23/2009
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