linking food science to biotechnology: unlock the color of ... · agarose is a polysaccharide...

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

Linking Food Science to Biotechology: Unlock the Color of Candies EDVO-Kit S-47

1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com

2

Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 1989-2015 EDVOTEK, Inc., all rights reserved. S-47.150909


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.

Linking Food Science to Biotechology: Unlock the Color of CandiesEDVO-Kit S-47

3




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



4



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

5




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

( - )


6



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


7




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


8



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.


OPTIONAL STOPPING POINTDyesamplesmaybestoredintherefrigeratorforupto24hoursbeforeperformingelectrophoresis.


9




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


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



10



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




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



12




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




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


14



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?

( - ) ( + )


15




Instructor's Guide


16


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.


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



INSTRUCTOR'S GUIDEEDVO-Kit S-47 Linking Food Science to Biotechology: Unlock the Color of Candies

Experiment Results and Analysis


18


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


20


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



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


22



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





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


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.


24





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




linking food science to biotechnology: unlock the color of ... · agarose is a polysaccharide...

Documents