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BasicBiotechnologyKit

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

Orange G, Xylene Cyanol,


Bromophenol Blue

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

wells in agarose gel


in center for dye electrophoresis

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

Polymerized agarose gel 1.5m scan size.


Digital Instruments

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.

Diagram of gel tray use screws on


side of tray to raise and lower gates. If
gates are not present, use lab tape to
seal ends.

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
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4. Whatdyeswereintheunknownmixture?

5. Whichnegativelychargeddyeseemstobethesmallestmolecule?

6. Listthedyesthathaveanegativecharge.

7. Inyourownwords,explainhowagarosegelelectrophoresisworks.

BiotechnologyintheClassroom2009
updated9/23/2009

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