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S-47.150909
S-47Edvo-Kit #S-47
Linking Food Science to Biotechnology: Unlock the Color of CandiesExperiment Objective:In this experiment, students will investigate how gel electrophoresis unlocks the color code by investigating food dyes used to make colorful candies.
See page 3 for storage instructions.
NEW
Page
Experiment Components 3
Experiment Requirements 3
Background Information: Candy Electrophoresis 4
Experiment Procedures Experiment Overview 6 Module I : Extraction of Food Dyes from Candy 9 Module II: Separation of Food Dyes by Agarose Gel Electrophoresis 10 Module III: STEM-Based Data Analysis of Food Dyes Using a Standard Curve 12 Study Questions 15 Instructor's Guidelines Overview of Instructor's Pre-Lab Preparations 16 Pre-Lab Preparations 17 Experiment Results and Analysis 18 Study Questions and Answers 19
Appendices 20 A EDVOTEK® Troubleshooting Guide 21 B Bulk Preparation of Agarose Gels 22 C Practice Gel Loading 23
Safety Data Sheets can be found on our website: www.edvotek.com
Table of Contents
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Linking Food Science to Biotechology: Unlock the Color of Candies EDVO-Kit S-47
Experiment Components
EDVOTEK and The Biotechnology Education Company are registered trademarks of EDVOTEK, Inc. Ready-to-Load, QuickStrips and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.
READY-TO-LOAD™ SAMPLES FOR ELECTROPHORESIS
Store all components at room temperature
Components Check (√)
A Dye Extraction Buffer qB Standard Dye Marker q
REAGENTS & SUPPLIES
• UltraSpec-Agarose™ q• Electrophoresisbuffer(50x) q• PracticeGelLoadingSolution q• Transfer pipet q• Calibrated transfer pipets q• PlasticCups q• 0.5mlMicrocentrifugeTubes q• 1.5 ml Microcentrifuge Tubes q
• Horizontalgelelectrophoresisapparatus• D.C.powersupply• Automaticmicropipetswithtips(optional)• Balance• Microwave,hotplateorburner• Pipetpump• 250mlflasksorbeakers• Hotgloves• Safetygogglesanddisposablelaboratorygloves• Visualizationsystem(whitelightbox)• Distilledordeionizedwater• Candies(suggestions:M&Ms®,Skittles®,JellyBeans,GumBalls)
All experiment components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.
Requirements
Experiment #S-47 is designed for 10 gels.
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Linking Food Science to Biotechology: Unlock the Color of CandiesEDVO-Kit S-47
Background Information: Candy Electrophoresis
FOOD COLORING
Thecoloroffoodhasalwaysbeenanintegralaspectofourculture.TheearlyRomansbelievedthatpeoplenotonlyeatwiththeirpalate,butalso“eatwiththeireyes.”Forcenturies,humanshaveuseddyesfromnaturalingredientstoaddcolortofood,drink,clothingandotherproducts.Forexample,saffron,paprikaandotherspiceswereusedtoprovidefoodyellowappearance.
Companieshavelongbeenintroducingcoloradditivestoavarietyofproducts,includingcandies,shampoos,perfumes,drinks,etc.Coloradditivesareusedinfoodforseveralreasons.Manufacturersaddcolorstofoodtooffsetcolorlossduetoproductexposuretovariousenvironmentalconditions,suchaslight,air,andmoisture.Additionally,companiesoftenadddyestofoodproductslikebeverages,jellies,puddingandcondimentstomakethemlookmoreattractivetoconsumers.Table1illustratesthesevencommonlyusedfooddyesintheUnitedStates.Ofthesedyes,Blue1andRed40are the most common blue and red dyes while Green3andBlue2arerarelyused.Foodcontain-ingtheseapproveddyesarecalled“ForColoringFood”andhavetheabbreviation“FCF”precedingtheirnames.Companiesarerequiredtolistfooddyesintheirlistofingredients(Figure1).
Astheusecontinuestogrow,concernsregardingtheadditionoffoodcolorstofoodproductsalsoemerge.Commonlyusedfooddyessuchasyellow5andred40arebelievetoposeseveralhealthconcernsinchildren,includinghyperactivityandallergicreactions.TheFoodandDrugAdministration(FDA)isresponsibleforregulatingcoloradditivesusedinavarietyofproductsintheUnitedStates.Coloradditivesallowedforuseinfoodsareclassifiedas“certifiable”or“exemptfromcertification”.CertifiablecoloradditivesaremanmadewhicharetestedbythemanufacturersandtheFDAtoassuresthequalityandsafetyofthecoloradditives.Ontheotherhand,coloradditivesthatare“exemptfromcertification”arethosethatderivedfromnaturalsourcessuchasvegetables,fruits,andminerals.
AGAROSE GEL ELECTROPHORESIS
Agarosegelelectrophoresisiswidelyusedtoseparatemoleculesbaseduponcharge,sizeandshape.ItisparticularlyusefulinseparatingchargedbiomoleculessuchasDNA,RNAandproteins.
Agarosegelelectrophoresispossessesgreatresolvingpower,yetisrelativelysimpleandstraightforwardtoperform.Thegelismadebydissolvingagarosepowderintheelectrophoresisbuffer.Thesolutionisboiledtodissolvetheagaroseandthencooledtoapproximately60ºCandpouredintoageltraywhereitsolidifies.Thetrayissubmergedinabuffer-filledelectrophoresisapparatus,whichcontainselectrodes.
Samples are prepared for electrophoresis by mixing them with glycerol or sucrose to give the mixture higher density.Thismakesthesamplesdenserthantheelectrophoresisbuffer.Thesesamplescanthenbeloadedwithamicropipetortransferpipetintowellsthatwerecreatedinthegelbyatemplateduringcasting.Thedensesamplessinkthroughthebufferandremaininthewells.
Figure 1 – Example of Candy Ingredient Label
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Abbreviation
Blue 1
Name Shade Structure
Blue 2
Green 3
Red 3
Red 40
Yellow 5
Brilliant Blue Blue
Indigo
Turquoise
Indigotine
Fast Green
PinkErythrosine
Allura Red Red
Tartazine Yellow
Yellow 6 Sunset Yellow Orange
Table 1 – Seven Artificial Colors Approved by the FDA for Coloring Food
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Adirectcurrentpowersupplyisconnectedtotheelectrophoresisapparatusandcurrentisapplied.Chargedmoleculesinthesampleenterthegelmatrix.Moleculeshavinganetnegativechargemigratetowardsthepositiveelectrode(anode)whilenetpositivelychargedmoleculesmigratetowardsthenegativeelectrode(cathode).Withinarange,thehighertheappliedvoltage,thefasterthesamplesmigrate.ThebufferservesasaconductorofelectricityandtocontrolthepH.ThepHisimportanttothechargeandstabilityofbiologicalmolecules.
Agaroseisapolysaccharidederivedfromagar.Inthisexperiment,UltraSpec-Agarose™,amixtureofagaroseandhydrocolloidswhichrendersthegeltobebothclearandresilient,isused.Atfirstglance,anagarosegelappearstobeasolidatroomtemperature.However,onthemolecularlevel,thegelcontainsmicroscopicporeswhichactasamolecularsieve,allowingthedifferentmoleculestopassthrough.
Fooddyesarecomposedofions.Whenthesechargedionsaresubjectedtoanelectricfield,themoleculeswillmigratetowardtheelectrodeofoppositecharge.Positivelychargedmoleculeswillmigratetowardthenegativeelectrode,whilethosewithanegativechargewillmovetowardthepositiveelectrode.Smalldyefragmentsmovethroughtheseholeseasily,butlargedyefragmentshaveamoredifficulttimesqueezingthroughthetunnels.
Factorssuchascharge,sizeandshape,togetherwithbufferconditions,gelconcentrationsandvoltage,affectsthemobilityofmoleculesingels.Becausemoleculeswithdissimilarsizestravelatdifferentspeeds,theybecomeseparatedandformdiscrete“bands”withinthegel.Afterthecurrentisstopped,thebandscanbevisualized(Figure2).
Inthisexperiment,studentswillextractseveraldifferentdyesfromfoodsource.Thedyeswillthenbeanalyzedusingagarosegelelectrophoresisandtheirrateofmigrationwillbeobservedandmeasured.
Figure 2 – Overview of agarose gel electrophoresis
( - )
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Candy Electrophoresis
EXPERIMENT OBJECTIVE:
Inthisexperiment,studentswillinvestigatehowgelelectrophoresisunlocksthecolorcodebyinvestigatingfooddyesusedtomakecolorfulcandies.
LABORATORY SAFETY
1. Glovesandgogglesshouldbewornroutinelyasgoodlaboratorypractice.2. Exerciseextremecautionwhenworkingwithequipmentthatisusedinconjunction
withtheheatingand/ormeltingofreagents.3. DONOTMOUTHPIPETREAGENTS-USEPIPETPUMPS. 4. Exercisecautionwhenusinganyelectricalequipmentinthelaboratory.5. Alwayswashhandsthoroughlywithsoapandwaterafterhandlingreagentsor
biologicalmaterialsinthelaboratory.
LABORATORY NOTEBOOKS:
Scientistsdocumenteverythingthathappensduringanexperiment,includingexperimentalconditions,thoughtsandobservationswhileconductingtheexperiment,and,ofcourse,anydatacollected.Today,you’llbedocument-ingyourexperimentinalaboratorynotebookoronaseparateworksheet.
Before starting the Experiment: • Carefullyreadtheintroductionandtheprotocol.Usethisinformationtoformahypothesisforthis
experiment. • Predicttheresultsofyourexperiment.
During the Experiment: • Recordyourobservations.
After the Experiment: • Interprettheresults–doesyourdatasupportorcontradictyourhypothesis? • Ifyourepeatedthisexperiment,whatwouldyouchange?Reviseyourhypothesistoreflectthischange.
Experiment Overview
Wear gloves and safety goggles
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Experiment Overview
After electrophoresis, transfer gel for visualization.
Analysis on white
light source
Attach safety cover,connect leads to power
source and conduct electrophoresis
Remove end blocks & comb, then submerge
gel under buffer in electrophoresis
chamber
Prepare agarose gel in
casting tray
6
5
3
2
( - )
Load eachdye sample inconsecutive wells
4
Prepare dye samples byextracting food dyesfrom candies
1
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Module I: Extraction of Food Dyes from Candy
T.C.
red
T.C.
blue
T.C.
yell
T.C.
T.C.
purT.C.
gre
1. 2. 3. 4.250 µlDye
ExtractionBuffer
Swirl
5. 6.
T.C.
red
7. 8.Rinse your cup.Repeat Steps 2-6.
T.C.
red
T.C.
blue
T.C.
yell
T.C.
gre
T.C.
pur
T.C.
We recommend using brightly-colored candies M&M's®, Skittles®, jelly beans, and gum balls
1. LABEL five microcentrifuge tubes with your initials and the colors of the candy you will beinvestigating.
2. LABELtheprovidedcupwithyourinitials. ADD one candy to the cup3. ADD 250µlofDyeExtractionBuffertothecupcontainingthecandy.4. SWIRL the candy gently in the Dye Extraction Buffer to dissolve the color coating until
thewhitelayerofthecandyisexposed.5. REMOVEthecandyfromthecup.6. TRANSFERthedissolvedcolorsolutionintotheappropriatelylabeledmicrocentrifugetube.7. RINSE thecup. REPEAT steps2-6withtheremaining4candies.8. PLACE thetubesonlabbench. PROCEED to Module II: Separation of Food Dyes by Agarose Gel Electrophoresis.
Wear gloves and safety goggles
OPTIONAL STOPPING POINTDyesamplesmaybestoredintherefrigeratorforupto24hoursbeforeperformingelectrophoresis.
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Linking Food Science to Biotechology: Unlock the Color of CandiesEDVO-Kit S-47
1. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A).2. MIX agarose powder with 1X buffer in a 250 ml flask (see Table A).3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Carefully REMOVE the flask from the microwave and MIX by swirling the flask. Continue to HEAT the solution in 15-second bursts until the agarose is completely dissolved (the solution should be clear like water).4. COOL agarose to 60° C with careful swirling to promote even dissipation of heat.5. While agarose is cooling, SEAL the ends of the gel-casting tray with the rubber end caps. PLACE the well template (comb) in the appropriate notch.6. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify within 20 minutes. The gel will stiffen and become less transparent as it solidifies.7. REMOVE end caps and comb. Take particular care when removing the comb to prevent damage to the wells.
60°C
1:001. 3.
4. 5.
7.
Caution! Flask will be HOT!
Concentratedbuffer
Distilledwater
Agarose
2.50x
Flask
© 2013 Edvotek® All Rights Reserved.
ConcentratedBuffer (50x)
Size of GelCasting tray
7 x 7 cm
7 x 10 cm
7 x 14 cm
0.6 ml
1.0 ml
1.2 ml
+DistilledWater
29.4 ml
49.0 ml
58.8 ml
+TOTALVolume
30 ml
50 ml
60 ml
=
Individual 0.8% UltraSpec-Agarose™ GelTable
A
60°C20min.
WAIT6.
Pour
Amt ofAgarose
0.23 g
0.39 g
0.46 g
Module II: Separation of Food Dyes by Agarose Gel Electrophoresis
1. DILUTEconcentrated(50X)bufferwithdistilledwatertocreate1Xbuffer(seeTableA).2. MIXagarosepowderwith1Xbufferina250mlflask(seeTableA).3. DISSOLVEagarosepowderbyboilingthesolution.MICROWAVEthesolutiononhighfor1minute.
Carefully REMOVEtheflaskfromthemicrowaveandMIXbyswirlingtheflask.ContinuetoHEAT the solution in15-secondburstsuntiltheagaroseiscompletelydissolved(thesolutionshouldbeclearlikewater).
4. COOLagaroseto60°Cwithcarefulswirlingtopromoteevendissipationofheat.5. Whileagaroseiscooling,SEALtheendsofthegel-castingtraywiththerubberendcaps.PLACE the well
template(comb)intheappropriatenotch.6. POUR the cooled agarose solution into the prepared
gel-castingtray.Thegelshouldthoroughlysolidifywithin20minutes.Thegelwillstiffenandbecomelesstransparentasitsolidifies.
7. REMOVEendcapsandcomb.Takeparticularcarewhenremovingthecombtopreventdamagetothewells.
IMPORTANT:
If you are unfamiliar with agarose gel prep and electrophoresis, detailed instructions and helpful resources are available at www.edvotek.com
Wear gloves and safety goggles
ConcentratedBuffer (50x)
Size of GelCasting tray
7 x 7 cm
7 x 10 cm
7 x 14 cm
0.6 ml
1.0 ml
1.2 ml
+DistilledWater
29.4 ml
49.0 ml
58.8 ml
+TOTALVolume
30 ml
50 ml
60 ml
=
Individual 0.8% UltraSpec-Agarose™ Gel
Amt ofAgarose
0.23 g
0.39 g
0.46 g
Table
A
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Module II: Separation of Food Dyes by Agarose Gel Electrophoresis
1X DilutedBuffer
8. 9.
10. 11. 12.( - )
( + )
1 2 3 4 5 6
Pour
Reminders:
If unfamiliar with gel loading, consider performing the optional activity in Appendix C, Practice Gel Loading, prior to performing the experiment.
Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.
8. PLACEgel(onthetray)intoelectrophoresischamber.COVERthegelwith1Xelectrophoresisbuffer(SeeTableBforrecommendedvolumes).Thegelshouldbecompletelysubmerged.
9. LOAD35µLoftheStandardDyeMarkerintothefirstwell.LOAD the remaining dye samples into the other fivelanes.BesuretoRECORDthecolorofeachsampleanditspositioninTable1.
10.PLACEsafetycover.CHECKthatthegelisproperlyoriented. Remember,thenegatively-chargedsampleswillmigratetowardthepositive(red)electrode.
11.CONNECT leads to the power source and PERFORM electrophoresis(SeeTableCfortimeandvoltageguidelines).12. Afterelectrophoresisiscomplete,REMOVE the gel and casting
tray from the electrophoresis chamber and VISUALIZE theresults.Nostainingisnecessary.
Lane
1
2
3
4
5
6
Table 2: Gel Loading
Candy Color
Standard Dye Marker
Time and Voltage Guidelines(0.8% Agarose Gel)
Min. / Max.Volts
150
125
75
15/20 min.
20/30 min.
35 / 45 min.
Table
CElectrophoresis Model
M6+M12 (classic)
& M36Min. / Max.
20/30 min.
30/35 min.
55/70 min.
M12 (new)
Min. / Max.
25 / 35 min.
35 / 45 min.
60 / 90 min.
50x Conc.Buffer
DistilledWater+
EDVOTEKModel #
Total Volume Required
1x Electrophoresis Buffer (Chamber Buffer)
M6+ & M12 (new)
M12 (classic)
M36
300 ml
400 ml
1000 ml
Dilution
Table
B
6 ml
8 ml
20 ml
294 ml
392 ml
980 ml
11
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Module III: STEM-Based Data Analysis of Food Dyes Using a Standard Curve
Agarose gel electrophoresis separates biomolecules intodiscretebands,eachcomprisingmoleculesofthesamesize.Howcantheseresultsbeusedtodeterminethelengthsofdifferentfragments?Remember,asthelengthofabiomoleculeincreases,the distance to which the molecule can migrate decreases because large molecules cannot pass throughthechannelsinthegelwithease.Therefore,the migration rate is inversely proportional to the lengthofthemolecules—morespecifically,tothelog10ofmolecule'ssize.Toillustratethis,weranasample that contains bands of known lengths called a “standard”.Wewillmeasurethedistancethateachofthesebandstraveledtocreateagraph,knownasa“standardcurve”,whichcanthenbeusedtoextrapolatethesizeofunknownmolecule(s).
1. Measure and Record Migration Distances
Measure the distance traveled by each Standard Dye Molecule from the lower edge of the sample well tothelowerendofeachband.Recordthedistanceincentimeters(tothenearestmillimeter)inyournotebook.Repeatthisforeachdyefragmentinthestandard.
Measure and record the migration distances of each of the fragments in the unknown samples in the same wayyoumeasuredthestandardbands.
2. Generate a Standard Curve.
Because migration rate is inversely proportional to the log10ofbandlength,plottingthedataasasemi-log plot will produce a straight line and allow ustoanalyzeanexponentialrangeoffragmentsizes.You will notice that the vertical axis of the semi-log plot appears atypical at first; the distance between numbersshrinksastheaxisprogressesfrom1to9.Thisisbecausetheaxisrepresentsalogarithmicscale.The first cycle on the y-axis corresponds to lengths from100-1,000basepairs,thesecondcyclemeasures1,000-10,000basepairs,andsoon.Tocreateastandardcurveonthesemi-logpaper,plotthedistance each Standard Dye Molecule migrated on the x-axis(inmm)versusitssizeonthey-axis(inbasepairs).Besuretolabeltheaxes!
A B C
Figure 3:Measure distance migrated from the lower edge of the well to the lower edge of each band.
Quick Reference:
The Standard dyes have the following base pair equivalents.
Blue 5000Red 3000Purple 1000Orange 500
Figure 4:Semilog graph example
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Module III: STEM-Based Data Analysis of Food Dyes Using a Standard Curve
Afterallthepointshavebeenplotted,usearulerorastraightedgetodrawthebeststraightlinepossiblethroughthepoints.Thelineshouldhaveapproximatelyequalnumbersofpointsscatteredoneachsideoftheline.Itisokayifthelinerunsthroughsomepoints(seeFigure4foranexample).
3. Determine the length of each unknown fragment.
a. Locatethemigrationdistanceoftheunknowndyeonthex-axisofyoursemi-loggraph.Drawa verticallineextendingfromthatpointuntilitintersectsthelineofyourstandardcurve.
b. Fromthepointofintersection,drawasecondline,thistimehorizontally,towardthey-axis.Thevalueatwhichthislineintersectsthey-axisrep-resentstheapproximatesizeofthedyeinbasepairs(refertoFigure4foranexample).Makenoteofthisinyourlabnotebook.
c. Repeatforeachfragmentinyourunknownsample.
Quick Reference:
Standard Dye marker sizes - length is expressed in base pairs.
5000, 3000, 1000, 500
13
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8,000
10,000
7,000 6,000
5,000
4,000
3,000
2,000
9,000
80 70 60
50
40
30
20
10
90 100
1,000
800 700 600
500
400
300
200
900
X-axis: Migration distance (cm)
1 cm 2 cm 3 cm 4 cm 5 cm 6 cm
Y-a
xis:
Ba
se P
airs
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Study Questions
1. Whatisthepurposeofgelelectrophoresis?
2. Whatdoyouthinkaresomefactorsthatdeterminethedistancemoleculesmovethoughthegel?Giveanexample.
3. IfyouwerepouringageltorunDNAsamples,wherewouldyouplacethecomb?Explain.
4. Thediagrambelowrepresentsyourgel.Usingcoloredpencilsormarkersdrawintheresultsofyourdyeseparation.Besuretolabeleachlanewithnameofthedyethatwasinthewell.
5. Whichdyewasthesmallestdyemolecule?Explain.
6. Whichdyewasthelargestdyemolecule?Explain.
7. WhendeterminingthesizesofDyefragments,whichaxisisusedtoplotthemigrationdistancesoftheknownandunknownfragments?Whichaxisisusedtoplotthesizesoftheknownandunknown
fragments?
( - ) ( + )
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Instructor's Guide
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INSTRUCTOR'S GUIDE Linking Food Science to Biotechology: Unlock the Color of Candies EDVO-Kit S-47
OVERVIEW OF INSTRUCTOR’S PRELAB PREPARATION:
This section outlines the recommended prelab preparations and approximate time requirement to complete each prelabactivity.
Preparation For: What to do: When: Time Required:
Module II: Separation of FoodDyes by Agarose Gel Electrophoresis
Prepare diluted TAE buffer
Prepare molten agarose and pour gel
40 min.
Module I: Extraction of FoodDyes from Candy
Aliquot Dye ExtractionBuffer
Aliquot Standard Dye Marker
Up to one day before performingthe experiment.
Up to one day before performingthe experiment.
Before the class period.
10 min.
Module III: STEM-Based Data Analysis of Food Dyes Using a Standard Curve
Photocopy/printsemi-log paper
10 min.
Pre-Lab Preparations:
For Module I, each group should receive the following materials:
• 1 tube Extraction Buffer• 1 plastic cup• 5 small microcentrifuge tubes• 1 small transfer pipet
NOTE:Accurate pipetting is critical for maximizing successful experi-ment results.
If students are unfamiliar with using micropipets, we recom-mend performing the optional activity found in Appendix C, Practice Gel Loading, prior to conducting the experiment.
MODULE I: EXTRACTION OF FOOD DYES FROM CANDY
•Aliquot1.3mlofExtractionBufferinto10labeledmicrocentrifugetubes.Distribute1tubeperstudentgroup.
MODULE II: SEPARATION OF FOOD DYES BY AGAROSE GEL ELECTROPHORESIS
Preparation of Agarose Gels
Thisexperimentrequiresone0.8%agarosegelperstudentgroup.A7x7cmgelisrecommended.Youcanchoosewhethertopreparethegelsinadvanceorhavethestudentspreparetheirown.Allowapproximately30-40minutesforthisprocedure.
• Individual Gel Preparation: Each student group can be responsible for casting their own individual gel
priortoconductingtheexperiment.SeeModuleIIintheStudent’s ExperimentalProcedure.Studentswillneed50xconcentratedbuffer,dis-
tilledwaterandagarosepowder.
• Batch Gel Preparation: Tosavetime,alargerquantityofagarosesolutioncanbepreparedfor sharingbytheclass.SeeAppendixB.
• Preparing Gels in Advance: Gelsmaybepreparedaheadandstoredforlateruse.Solidifiedgelscanbe
storeunderbufferintherefrigeratorforupto2weeks.Donotfreezegelsat-20ºCasfreezingwilldestroythegels.
Gels that have been removed from their trays for storage should be “anchored”backtothetraywithafewdropsofmoltenagarosebefore
beingplacedintothetray.Thiswillpreventthegelsfromslidingaroundinthetraysandthechambers.
Additional Materials:
Each0.8%gelshouldbeloadedwiththeStandardDyeMarkerand5samplescontainingdissolvedcoloredcandycoating.
•Aliquot40µloftheStandardDyeMarker(comp.B)intolabeledmicrocentrifugetubesanddistributeonetubeofladderperstudentgroup.
MODULE III: STEM-BASED DATA ANALYSIS OF FOOD DYES USING A STANDARD CURVE
Preparecopiesofsemi-logpaperanddistributeonecopyperstudentgroup.
For Module II, each group should receive the following materials:• 50x concentrated buffer• Distilled Water • UltraSpec-Agarose™• Candy dye samples
17
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INSTRUCTOR'S GUIDEEDVO-Kit S-47 Linking Food Science to Biotechology: Unlock the Color of Candies
Experiment Results and Analysis
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INSTRUCTOR'S GUIDE Linking Food Science to Biotechology: Unlock the Color of Candies EDVO-Kit S-47
Lane Sample1 Standard Dye Marker2 Dye extracted from Candy #13 Dye extracted from Candy #24 Dye extracted from Candy #35 Dye extracted from Candy #46 Dye extracted from Candy #5
1 2 3 4 5 6 1 2 3 4 5 6
NOTE: In the idealized schematic, the relative positions of dye fragments are shown but are not depicted to scale. No positively charged dyes are shown.
Quick Reference:
Standard Dye marker sizes - length is expressed in base pairs.
5000, 3000, 1000, 500
Please refer to the kit insert for the Answers to
Study Questions
A EDVOTEK® Troubleshooting Guide
B Bulk Preparation of Agarose Gels
C Practice Gel Loading
Safety Data Sheets can be found on our website: www.edvotek.com
Appendices
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APPENDICES Linking Food Science to Biotechology: Unlock the Color of Candies EDVO-Kit S-47
Appendix AEDVOTEK® Troubleshooting Guides
PROBLEM: CAUSE: ANSWER:
The dye sample was very light after the extraction procedure.
Try dissolving the dye off of multiple candies of the same color.Not enough candy used for extraction.
Very light colored band seen after electrophoresis
Pipetting error Make sure students pipet 35 µl of dissolved colored candy coating per well.
The gel was not prepared properly. Ensure that the electrophoresis buffer was correctly diluted.
Malfunctioning electrophoresis unit or power source.
Contact the manufacturer of the electrophoresis unit or power source.
I was not able to extract color from my food samples.
Not enough food sample used for extraction.
Try dissolving the dye off of multiple candies of the same color. Also be sure that the waxy coating, if any, is dissolved.
21
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APPENDICESEDVO-Kit S-47 Linking Food Science to Biotechology: Unlock the Color of Candies
Appendix B
Tosavetime,theelectrophoresisbufferandagarosegelsolutioncanbepreparedinlargerquantitiesforsharingbytheclass.Unuseddilutedbuffercanbeusedatalatertimeandsolidifiedagarosegelsolutioncanberemelted.
Bulk Preparation of Agarose Gels
Bulk Electrophoresis Buffer
Quantity(bulk)preparationfor3litersof1xelectro-phoresisbufferisoutlinedinTableD.
Batch Agarose Gels (0.8%)
Forquantity(batch)preparationof0.8%agarosegels,seeTableE.
1. Usea500mlflasktopreparethedilutedgelbuffer
2. Pour3.0gramsofUltraSpec-Agarose™intothepreparedbuffer.Swirltodisperseclumps.
3. Withamarkingpen,indicatethelevelofsolutionvolumeontheoutsideoftheflask.
4. Heattheagarosesolutionasoutlinedpreviouslyforindividualgelpreparation.Theheatingtimewillrequireadjustmentduetothelargertotalvolumeofgelbuffersolution.
5. Cooltheagarosesolutionto60°Cwithswirlingtopromoteevendissipationofheat.Ifevaporationhasoccurred,adddistilledwatertobringthesolu-tionuptotheoriginalvolumeasmarkedontheflaskinstep3.
6. Dispensetherequiredvolumeofcooledagarosesolutionforcastingeachgel.ThevolumerequiredisdependentuponthesizeofthegelbedandDNAstainingmethodwhichwillbeused.RefertoAppendixAorBforguidelines.
7. Allowthegeltocompletelysolidify.Itwillbecome firm and cool to the touch after approxi-mately20minutes.Thenproceedwithpreparingthegelforelectrophoresis.
60˚C
Note: The UltraSpec-Agarose™ kit component is usually labeled with the amount it contains. Please read the label care-fully. If the amount of aga-rose is not specified or if the bottle's plastic seal has been broken, weigh the agarose to ensure you are using the correct amount.
50x Conc.Buffer +
DistilledWater
Total Volume Required
60 ml 2,940 ml 3000 ml (3 L)
Bulk Preparation of Electrophoresis BufferTable
D
Batch Prep of 0.8% UltraSpec-Agarose™Table
EAmt ofAgarose
(g)
ConcentratedBuffer (50X)
(ml)+
DistilledWater(ml)
TotalVolume
(ml)+
3.0 7.5 382.5 390
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APPENDICES Linking Food Science to Biotechology: Unlock the Color of Candies EDVO-Kit S-47
Appendix CPractice Gel Loading
Accuratesampledeliverytechniqueensuresthebestpossiblegelresults.Pipettingmistakescancausethesampletobecomedilutedwithbuffer,orcausedamagetothewellswiththepipettipwhileloadingthegel.
Ifyouareunfamiliarwithloadingsamplesinagarosegels,itisrecommendedthatyoupracticesampledeliverytech-niquesbeforeconductingtheactualexperiment.EDVOTEKelectrophoresisexperimentscontainatubeofpracticegelloadingsolutionforthispurpose.Castingofaseparatepracticegelishighlyrecommended.Onesuggestedactivityisoutlined below:
1. Castagelwiththemaximumnumberofwellspossible.
2. Afterthegelsolidifies,placeitunderbufferinanelectrophoresisapparatuscham-ber.
Alternatively,yourteachermayhavecutthegelinsectionsbetweentherowsof
wells.Placeagelsectionwithwellsintoasmall,shallowtrayandsubmergeitunderbufferorwater.
3. Practicedeliveringthepracticegelloadingsolutiontothesamplewells.Takecarenottodamageorpuncturethewellswiththepipettip.
• Forelectrophoresisofdyes,loadthesamplewellwith35-38microli-tersofsample.
• Ifusingtransferpipetsforsampledelivery,loadeachsamplewelluntilitisfull.
4. Ifyouneedmorepractice,removethepracticegelloadingsolutionbysquirtingbufferintothewellswithatransferpipet.
5. Replacethepracticegelwithafreshgelfortheactualexperiment.Note: If practicing gel loading in the electrophoresis chamber, the practice gel loading solution will become diluted in the buffer in the apparatus. It will not interfere with the experiment, so it is not necessary to prepare fresh buffer.
Note: The agarose gel is some-times called a "submarine gel" because it is submerged under buffer for sample loading and electrophoretic separation.
Note: If you do not wish to pour extra agarosegels,Edvotek®DuraGels™(Cat.S-43)canbeusedasasub-stitute.Edvotek®DuraGels™arereusable polymer gel models that allows students to gain experience with gel loading before performing agarosegelelectrophoresis.TheuseofDuraGels™eliminatesthepreparationtime,expense,andwaste of pouring actual agarose practicegels.
23
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APPENDICESEDVO-Kit S-47 Linking Food Science to Biotechology: Unlock the Color of Candies
Appendix CPractice Gel Loading
2-20 µl
20-200 µl
100-1000 µl
2 0.0
2 – 20 µl
tens, ones, tenths (in decimal)
2 0.0
20 – 200 µl
2 0 0hundreds, tens, ones
100 – 1000 µl
1 0 0 0thousands, hundreds, tens, ones
2 0 0
1 0 0 0
© All rights reserved, Edvotek, Inc. 2013
Wear gloves and safety goggles
SETTING THE VOLUME OF AN ADJUSTABLE VOLUME MICROPIPET
1. CHOOSEthecorrectmicropipetforthevolumeyouaremeasuring.MakesurethatthevolumetobemeasuredDOES NOT EXCEEDtheupperorlowervolumesettingofthemicropipet.
2. DETERMINEtheunitsmeasuredbythemicropipetbylookingatthevolumesetting.Thesettingwillappearinthewindowonthesideofthemicropipet.Notethatthedifferentmicropipetsusedifferentscalesfortheirmeasure-ments.Somemicropipetsareaccuratetoatenthofamicroliter,whileothersareaccuratetoonemicroliter.
3. SETthevolumebytwistingthetopoftheplunger.Ingeneral,twistingtheplungerclockwisereducesthevolume,andtwistingtheplungercounterclockwiseincreasesthevolume.
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APPENDICES Linking Food Science to Biotechology: Unlock the Color of Candies EDVO-Kit S-47
Appendix CPractice Gel Loading
Wear gloves and safety goggles
MEASURING LIQUIDS WITH A MICROPIPET
1. SETthemicropipettotheappropriatevolumebyadjustingthedial.
2. PLACEacleantiponthemicropipet.
3. PRESStheplungerdowntothefirststop.HOLD the plunger down while placing the tip beneaththesurfaceoftheliquid.
4. SlowlyRELEASEtheplungertodrawsampleintothepipettetip.Positionthepipettipoverthewell.Becarefulnottopunctureordamagethewellwiththepipettip.
5. DELIVERthesamplebyslowlypressingtheplungertothefirststop.Depresstheplungertothesecondstoptoexpelanyremainingsample.DO NOT RELEASE the plunger until the tipisoutofthebuffer.
6. DISCARD thetipbypressingtheejectorbutton.Useanewcleantipforthenextsample.
4 5 6Dial
1 2 3
2 0.0
2 0
.0
2 0
.0
2 0
.0
2 0
.0
2 0
.0
2 0
.0
25
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APPENDICESEDVO-Kit S-47 Linking Food Science to Biotechology: Unlock the Color of Candies
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