Molecular Scissors:
Lambda Digest
Student Materials
Introduction…………………………………………………………………………………………………………………………2
Pre-LabQuestions……………………………………………………………………………………………………………….5
LabProtocol…………………………………………………………………………………………………………………………6
DataCollectionWorksheet………………………………………………………………………………………………….9
Post-LabQuestionsandAnalysis………………………………………………………………………………………..10
PlasmidMaps…………………………………………………………………………………………………………………….13
Lastupdated:3/15/2017
Molecular Scissors
Introduction
Doyouknowwhatrestrictionenzymesare?Alsoknownasrestrictionendonucleases,restriction
enzymesareimportantenzymesthatcutDNAatspecificsequencescalledrestrictionsites.Restriction
enzymeswerediscoveredwhenscientistsnoticedthatbacterialviruses(bacteriophages)caninfect
somebacterialcellsandnotothers.Thebacterialhostcellsthatareresistanttoinfectionproduce
enzymesthatcuttheinvadingviralDNA.ThesmallfragmentsofviralDNAcannotbeusedtomakenew
completeviruses–stoppinginfectionofthebacteriacell.Scientistnamedtheseenzymesrestriction
endonucleasesorrestrictionenzymesfortheirabilityto“restrict”orlimitviralinfection(seeFigure1).
Figure1.Bacteriadefendingthemslevesagainstbacteriophages
http://www.biomedheads.com/uploads/2/6/0/4/26041745/8703912_orig.jpg
Sincethediscoveryofthefirstrestrictionenzyme,hundredsofrestrictionenzymeseachthatrecognizes
andcutsDNAatdifferentrestrictionsiteshavebeenindentified.Thenameofeachrestrictionenzyme
comesfromthegenus,speciesandstrainofthebacteriathatnaturallyproducedit.Forexample,the
enzymeEcoRIcomesfrom:
E=Eschericiagenus
co=colispecies
R=strainRY13
I=romannumeralIforthefirstenzymeindentifiedinthebacterium
Mostoftherestrictionezymesusedbyscientistsrecognizerestrictionsitesthatarefourtoeightbase
pairslong.Let’slookatarestrictionsiteforEcoRI:
5’GAATTC3’
3’CTTAAG5’
Lookcloselyatthesequences.Doyounoticeanythingspecial?Readthetopstrandlefttoright
outloud….nowreadthebottomstrandrighttoleft.
2
Didyounoticethatyouwerereadingthesamesequence?Thisiscalledapalindromeanditisoftena
characteristicofrestrictionsites.Inthiscase,EcoRIcutsbetweentheGandtheAwhenreadingfrom5’
to3’.Becausetheenzymecutseachofthetwostrandsoftherestrictionsite,itleavesstaggeredcuts
thatproduce“stickyends.”Theseshort,unpairedsequencesarethesameforanyDNAthatiscutby
EcoRI.
Thisissometimeindicatedinthismanner:
5’G/AATTC3’ 5’GAATTC3’
or
3’CTTAA/G5’ à
5’ATCAGG
3’TAGTCC
CCATT3’
GGTAA5’
3’CTTAA
G5’
Whilenotascommon,someenzymeslikeHaeIIIcutbothstrandsofDNAatthesameposition,
producingbluntends.
5’ATCAGG/CCATT3’
3’TAGTCC/GGTAA5’
TheabilityofrestrictionenzymestocutDNAatknownsitesmakethemanimportanttoolin
biotechnology.ScientistscancutopenaDNAsequenceandinsertanothersequenceintotheopening
(Figure2).
Figure2.UsingrestrictionenzymestocreaterecombinantDNA
5’
3’
Originaldouble-strandedDNAmolecule
withasinglerestrictionsiteforEcoRI
3’
GAATTC
CTTAAG
FragmentsoforiginalDNAmolecule
producedbydigestionwithEcoRIand
double-strandedDNAtobeinserted
intooriginalDNAmolecule.
restriction
enzymesite
Cutwithrestrictionenzyme
5’
3’
NewDNAcomposedoforiginal
moleculeandinsertedmolecule.
5’
5’
3’
3’
5’
AATTC
G
CTTAAG
5’AATTC
G
3’
G 3’
CTTAA 5’
G AATTC
CTTAA
G
JoinDNAfragmentswithDNAligase
3
’5
’
GAATTC
G
CTTAA
Whydoyouthinkthisissuchanimportantprocedure?
IfyouthoughtaboutinsertingDNAfromoneorganismintotheanotherorganisms,ormakingcertain
drugs,orfixingageneticdiseasethenyouareontherighttrack.Forexample,ifascientistwantsto
studyaspecificprotein,shecaninsertthegeneforthatproteinintobacterialDNAandhavemillionsof
bacterialcellsmaketheprotein.Itisnotreallyquitethatsimplebutyougettheidea.
RestrictionenzymesalsoallowscientiststocreatemapsofDNAbasedonthepostionofrestrictionsites.
OnekindofphysicalmapofDNAisarestrictionmap.Restrictionmapsincludethelocationandorderof
3
sitescutbydifferentrestrictionenzymes(seeFigure3).Thesephysicalmapsareimportanttoscientists
astheyidentifiyandworkwithDNAmolecules.
Figure3.RestrictionmapoflambdaDNAcutwithBamHI
Fragmentsizes
5505
16,841
5626 6527
7233
6770
basepairs(bp)
Howdoyoucreatearestrictionmap?AftertheDNAiscutwithdifferentenzymes,scientistseparatethe
fragmentsbygelelectrophoresisandanalyzetheresultingpatterns.Gelelectrophoresisisacommon
waytomeasuremolecularsizes.Hereisananalogytoexplaingelelectrophoresis.Imagineaverydense
forestoftreeswhereeachtreeisonlyonefootawayfromeveryothertree.Youandamousemust
eachrunthroughtheforestfrompointAtopointB.Youandthemousebeginatthesametime.Which
willreachtheothersidefirst,youorthemouse?
Ingelelectrophoresis,thegelisanalogoustotheovergrownforest.Thegelisameshformedof
polymers,muchlikeJELL-O®.JELL-Oisameshofgelatinmadefromanimalproductslikecollagen.The
gelsforelectrophoresisareameshofagarose,whichispurifiedfromseaweed.Ingelelectrophoresis
themoleculesareanalogoustoyouandthemouse.Smallmoleculeswillmovemorequicklythrough
themeshthanwilllargemolecules,forthesamereasonsthatthemousemovesmorequicklythrough
thethickforestthanyoudo.
Sofarsogood,butonceamoleculeissuspendedinthegel,whydoesitmove?DNA,RNAandprotein
moleculesoftenhaveasurfacecharge.Thismeansthatifanelectricfieldisappliedtothegel,
moleculeswithanegativesurfacechargewillmovethroughthegeltowardsthepositivelycharged
anodeandmoleculeswithapositivesurfacechargewillmovethroughthegeltowardsthenegatively
chargedcathode.Whentwo(ormore)moleculeshavethesamechargethemoleculeswillmove
throughthegelbasedupontheirsize,smallermoleculesmovefasterthroughthegelthenlarger
molecules(Figure4).
Figure 4. Agarose gel electrophoresis of DNA
http://www.discoveryandinnovation.com/BIOL202/notes/lecture23.html
Inthislab,youwillbeingusingDNAfromabacteriophagecalledlambda(l)andcuttingitwithtwo
enzymes:EcoRIandHindIII.OnceyouhavecuttheDNA,youwillrunitonanelectrophoresisgeland
comparelambda(l)cutwiththetwodifferentenzymesanduncutlambda(l)DNA.
Tomakethingsalittlemoreinteresting,theenzymesarenotidentified.Therearetwotubes,AandB.
OnehasEcoRIandthetheotherhasHindIII.Yourjobistofigureoutwhichtubecontainswhichenzyme.
4
Molecular Scissors
Pre-LabQuestions
Directions:AfterreadingthroughtheintroductionandprotocolfortheMolecularScissorslab,answer
thefollowingquestions.
1. Whatarerestrictionenzymesandwherearetheyfoundinnature?
2. YoufindatypeofbacteriathatisresistanttotheT-4bacteriophage.Howmightyouexplainthe
bacterium’sresistancetothisvirus?
3. TherestrictionenzymePvuIrecognizesa6basepair,“palindromic”sequenceindoublestranded
DNA.Threebasesofonestrandaregiven,completetherestrictionsiteforPvuI.
5’
3’
T↓CG 3’
5’
PvuIcutsbothstrandsofDNA.Thepositionofthefirstcutisindicatedbythearrowabove.
Drawanarrowtoindicatethepositionofthesecondcut.
4. ImagineyouhaveacircularpieceofbacterialDNA(aplasmid)inwhichyouwanttoinsertageneto
studyaprotein.Youneedtodecidewhatrestrictionenzymetousetocutopentheplasmid–EcoRI
orHaeIII.Whatenzymewouldyouusetocuttheplasmid?Explainyouranswer.
5. ManymoleculesofDNAfromavirushavebeencutwithvariouscombinationsoftherestriction
enzymesEcoRI,BamHI,HindIII.Therestrictionmapandresultinggelareshownbelow.Which
sampleonthegelshowsDNAthathasbeencutwithbothBamHIandHindIII?Explainyouranswer.
DNAfromvirus
EcoRI BamHI
1
2
3
4
wells
EcoRI
HindIII
5
Molecular Scissors
LabProtocol
There’sbeenamixupinthelab–labelsonyourtubesofrestrictionenzymeshavefallenoff.Youknow
thatyouhaveatubeofEcoRIandatubeofHindIII,butyoudon’tknowwhichoneiswhich.Inorderto
identifytheenzymesintubesAandB,youwillhavetoperformdigestsofDNAfromabacteriophage
calledlambda(l).OnceyouhavecuttheDNAyouwillrunitonanelectrophoresisgeltocomparethe
cutanduncutDNAandtheresultsofthetwodifferentenzymes.Bycomparingthegelresultstothe
restrictionsmapsinFigure5youwillbeabletofigurewhichtubecontainswhichenzyme.
Figure5:RestrictionMapsforLambda/HindIIIandLambda/EcoRI
Materials:checkyourworkstationstomakesureallsuppliesarepresentbeforebeginningthelab.
StudentWorkstation:
CommonWorkstation:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
1icebucketorstyrofoamcupwithcrushedice
1p20micropipetteandpipettetips
1microcentrifugetuberack
1tubewith500µLwater
1tubeCutSmart®bufferwith20µLbuffer
1tubelambdaDNAwith10µLDNA(500ng/µl)
1tubeEnzymeAwith1µLrestrictionenzyme
1tubeEnzymeBwith1µLrestrictionenzyme
1tubeDNALadderwith10µLQuickLoad®1kbExtendLadder
1tubeControl(empty)
1tubeloadingdyewith30µL6Xdye
1agarosegel(0.6%)withDNAstain
1electrophoresisunitwithpowersupply
1extrafinepointpermanentmarker
•
37°Cwaterbathorincubator
65°Cwaterbathorincubator
microcentrifuge(optional)
UVlightorbluelight
transilluminator
1Xelectrophoresisbuffer
6
Caution:Keepallreagenttubesonice.
Procedure:
1. Findthefourmicrocentrifugetubes,labeledenzA,enzB,ControlandLadder
2. Usingyourp20micropipette,setuptherestrictiondigestsusingTable1below.Itisagoodideato
checkoffthereagentsasyouaddthem.
Table1:Set-upforrestrictiondigestoflambdawithEcoRIandHindIII.
Addthe
Tube
FollowingReagents:
EnzA
EnzB
Control
Ladder
Water
42µL
42µL
43µL
5µL
CutSmartBuffer
none
5µL
5µL
5µL
none
lDNA
2µL
2µL
2µL
EnzymeA(alreadyadded)
none
none
none
1µL
EnzymeB(alreadyadded)
none
none
none
1µL
Ladder(alreadyadded)
none
none
none
10µL
50µL
50µL
50µL
15µL
TotalVolume
Caution:Makesureyouuseacleantipforeachpipettingtransfer.
3. Usingthecentrifuge,quicklyspinthetubestogetallthereagentstothebottom.Ifyoudonothave
acentrifuge,youcangentlytapthemonthebenchtoconsolidatethecontentsinthebottomofthe
tube.
4. PutalltubesexcepttheLadderinthe37°Cwaterbathfor30-45minutesorforthetimeittakesyou
tomakeyourgel.
5. Duringtheincubation,add5µLofsterilewatertothetubelabeledladder.
6. Prepareorsourcea0.6%agarosegeland1Xelectrophoresisbufferasinstructedbyyourteacher.
7. Removeyourreactiontubesfromthe37°Cwaterbath.
StoppingPoint–Checkwithyourteacherbeforecontinuingwiththeprotocol.
8. Add8µLofloadingdyetothereactiontubes(EnzA,EnzBandControl).Mixbyflickingthetubeand
poolreagentsatthebottombycentrifugingortappingonthebench.
Caution:Makesureyouuseacleantipforeachreactiontube.
9. Afterloadingdyeisadded,heatyourreactiontubesto65°Cfor5minutes.Movetubesdirectlyto
ice.
7
Caution:DonotheattheLadder.
10. Assemblethegelboxandpositionitwhereitwillrun.
Tip:Checktomakesurethatthegeltrayisinthecorrectionorientationwiththewellsclosest
tothenegativeelectrode.
11. Load15µLofeachsampleandtheladderintoawellofthegelandrecordwhichsamplewentinto
whichwell.UseTable2tohelpyouknowwhereonthegeleachsampleisloaded.
12. Runyoursamplesasinstructedbyyourteachersuntilthefrontloadingdyeistwo-thirdsoftheway
downthegel.
Table2:Gelelectrophoresis
Lane(leftto
right)
Sample
name
1
Caution:Makesureyouuseacleantipforeachsample.
2
3
4
5
6
7
8
13. Whilethegelisrunningcalculatethesizeoftherestrictionfragmentsthatyouexpectwhenlamda
DNAiscutwitheachoftheenzymesandrecordtheminTable3below.
Table3:SingledigestrestrictionfragmentsofLambdaDNAcutwithEcoRIandHindlll
• Hint:fortheHindIIIdigest,look
HindIII
EcoRI
atthemapinFigure5.Starting
at“start”andmovingtothefirst
Restriction
Fragmentsin
Restriction
Fragmentsin
restrictionsite,thesizeofthe
Fragments
descendingorder
Fragments
descendingorder
fragmentis23,130bp.
23,130
•
Todeterminethesizeofthe
2,027
secondfragment,subtractthe
basepairnumberofthefirstsite
(23,130)fromthenumberofthe
secondsite(25,157).
•
Continuethisprocess,recording
thesizeofthefragmentsinthe
table.Whenyouhavefinished,
inthesecondcolumnofthe
tablelistthefragmentsin
descendingorder.
14. Onceyourgelisfinishedrunningexaminethelocationofthebandsandcompletethedata
collectionworksheet.
8
Molecular Scissors
DataCollectionWorksheet
Directions:AftercompletingtheMolecularScissorslab,answerthequestionsbelow.
1. Ontheimagebelowdrawwhatyouseeaftergelelectrophoresis
−electrode
Ladder(Kb)
48.5
20
15
10
8
6
5
4
3
2
1.5
1
0.5
+electrode
2. DeterminewhichenzymewasEcoRIandwhichwasHindIII.Recorditbelow.
EnzymeA_______________
EnzymeB_______________
3. TherestrictionsiteforEcoRIisGAATTC.Basedonyourresults,howmanytimesdoesthesequence
occurinthelambdasequence?______
4. Whenyoucomparetherestrictionmapstoyourresultsarethereanymissingbands?Canyou
suggestanyreasonswhythereisthisdiscrepancy?
5. WhatisthetotallengthoftheLambdaDNA?
9
Molecular Scissors
Post-LabQuestionsandAnalysis
Directions:AftercompletingtheMolecularScissorslab,answerthequestionsbelow.
ScientistscanuseelectrophoresisresultstodeterminethesizeofunknownDNAsamples.Scientists
havedeterminedthatDNAfragmentsmovethroughthegelatatratethatisinverselyproportionalto
thelog10oftheirmolecularweight(ornumberofbasepairs).So,ifyourunasetoffragmentswith
knownsizesonagelalongwiththeunknownfragment,youcancreateastandardcurvewiththeknown
fragmentsanduseittodeterminethesizeoftheunknownfragments.Let’stryit.
1
2
3
A
4
5
B
C
1. ThegelimageabovedepictsagelwithaDNAstandardinlane1andtwounknownsinlanes3and5.
MeasurethedistancethateachbandintheDNAstandardhasmovedfromthewellandrecordit
nexttothecorrespondingbandinTable4(onthenextpage).Tip:Youshouldmeasurefromthe
frontedgeofthewelltotheleadingedge(furthestfromthewell)ofeachband
2. Measurethedistancethateachoftheunknownbandshastraveledfromthewellsandrecordthe
distanceinTable4.
3. UsethedatafromTable4tocreateastandardcurveusingeitherExcelorsemi-logpaper.Thexaxis
isdistancetraveledandtheyaxisisbasepairlength.Connectthedatapointswitha“best-fit”line.
4. Usethestandardcurvetodeterminethesizeofthethreeunknownfragments.Recordthesizeof
thefragmentsA,BandCinTable4.
10
Table4.Restrictiondigestgeloftwounknowns.
DNAStandard
Size(bp)ofDNA
Distance(mm)moved
fromwell
Skipthisband–verylarge
23,000
bandswillskewthestandard
curve
9,416
6,557
4,361
2,322
2,027
Unknowns
Band
Size(bp)of
Distance(mm)moved
DNA
fromwell
A
30.9
B
C
5. UsethedatafromTable4tocreateastandardcurveusingeitherExcelorsemi-logpaper.Thexaxis
isdistancetraveledandtheyaxisisbasepairlength.Connectthedatapointswitha“best-fit”line.
6. Usethestandardcurvetodeterminethesizeofthethreeunknownfragments.Recordthesizeof
thefragmentsA,BandCinTable4.
11
12
Maps of Lambda DNA with EcoRI and HindIII Restriction sites
13