SBI4U-Biotechnology
Date:________________________________________
GelElectrophoresisActivity
PARTA:PrinciplesofGelElectrophoresis
Directions:
1.GototheDNAiwebsitewww.dnai.org>Manipulation>Techniques>sortingandsequencing.
2.ViewtheGelElectrophoresis2-Danimation,andanswerthefollowingquestions.
Questions:
1.HowdoestheprocessofgelelectrophoresisseparateDNAfragments?
ItusesanelectriccurrenttoseparatedifferentsizedmoleculesofDNAinaporoussponge-likematrix.
2.a)Whatisthepurposeoftheagarosegel?
ToseparatethedifferentsizedfragmentsofDNA.
b)Whatwouldincreasingtheconcentrationofagarosehaveonyouranswerin2(a)?(Note:Answernotfoundon
thewebsite.)
AhigherconcentrationwouldimpedetheDNAfragmentstotravelwithinthegel.Thisprovidesbetter
separationoffragmentsthatarecloseinsizewitheachother–bestwithsmallfragments,notlargefragments.
3.Whatisthepurposeofaddingblue“tracking”dyetotheDNAsamples?
ItmakesiteasiertoloadthesamplesandvisuallytrackthemigrationofDNA
throughthegel.
4.ExplainwhyDNAhasanoverallnegativecharge.
PhosphategroupsintheDNAbackbonecarrynegatively-chargedoxygen
moleculesgivingthephosphate-sugarbackboneofDNAanoverallnegative
charge.
5.WhyisthefactthatDNAhasanegativechargesoimportantinthegel
electrophoresisprocess?
ThenegativelychargedDNAcanbepulledtowardthepositivefieldofthegel.
6.ExplainhowanagarosegelcanseparateDNAfragmentsofdifferentlengths.
Smallerfragmentsmovefaster,andthereforefurther,thanlargerfragmentsas
theysnakethroughthegel.Seediagramatrightforvisual.
7.Theagarosegelisimmersedinbuffersolution.Whatistheroleofthebuffersolution?HINT:Thinkelectrolytes.
Thebuffersolutionallowsfortheflowofanelectricalcurrentthroughthegel.
8.Whatisthepurposeofethidiumbromideingelelectrophoresis?
EthidiumbromideisadyeusedbyscientiststoseewheretheDNAfragmentsarelocatedinthegel.Itbindsto
theDNAandglowswhenilluminatedwithUVlight.Pleasenotewewillbeusingmethyleneblueasethidium
bromideisdeemedtobeacarcinogen.
8.Whyisamarkerusedwhenrunningthefragmentsthroughthegel?
AmarkercontainsDNAfragmentsofknownsize.Markersarerunineverygelforcomparisonwiththeunknown
fragmentsinothergellanes.TheDNAfragments’distancearemeasuredonthegelandthenplottedona
standardgraph(FragmentLengthinbpvs.DistanceMigratedinmm).
Thesizesoftheunknownfragmentsaredeterminedbymeasuringtheirdistancemigratedandinterpolatingthe
fragmentlengthusingthestandardizedgraph.Wewillbedoingthisinourlab.
9.Whatisarestrictionmap?
ItshowswhererestrictionenzymescuttheoriginalpieceofDNA.
On the gel picture below,
10.
10.Onthegelpicturebelow,
(a) circle the smallest fragment produced by a restriction enzyme and label it “smallest.”
(a)circlethesmallestfragmentproducedbyarestrictionenzymeandlabelit“smallest.”
(b) circle the largest fragment produced by a restriction enzyme and label it “largest.”
(b)circlethelargestfragmentproducedbyarestrictionenzymeandlabelit“largest.”
Largest
Smallest
11.Inoneortwosentences,summarizethetechniqueofgelelectrophor
esis.
11. In one or two sentences, summarize the technique of gel electrophoresis. Student answers
Nowthat’sforyoutoanswer.J
DNA restriction fragment size chart