PlasmidIdentificationConclusion

byHannahGay
Introduction
Aplasmidissmallcircular,doublestrandedDNAmolecule(Nature.com,n.d)thatarefound
naturallyinbacteriaandarebasicallyusedasvectors.(Nature.com,n.d)Thepurposeofthislabwasto
takeanunknownplasmidandfindoutwhichoneofthethreepossibleplasmidsitcouldbepAMP,
pKAN,orpBLU.ThiswastheperfectwaytoseeifIcoulduseallthenewskillsIlearnedoverthe
courseofthesemesterinonefinalassignment.
Ourbasicstrategywassimple.Firstwehadtochosewhichrestrictionenzymestouseforour
singleanddoubledigests.Restrictionenzymesmustbeused.RestrictionenzymesareDNAcutting
enzymes(RestrictionEnzymes2014)thatcuttheDNAintofragments.Then,wehadtocalculateall
theingredientsneededforcompletinggelelectrophoresis.Gelelectrophoresisistheprocessusedin
ordertoseparatemacromoleculeslikeDNA(BiotechLearningHub,n.d).Itsbasicallyagelmade
from0.8%agaroseand10wellstohold10differentsolutionsfortestingcovered1xTAEbufferwith.
Finally,oncewefinishedrunningthegelwehadtocreateastandardcurveinordertoaccurately
determinethesizesofourDNAfragments.Onceallofthatwasfinished,onlythenwouldwebeableto
accuratelydeterminewhichplasmidwehad.
Methods
BeforeImoveon,ImustmentionwhereallthematerialsIusedforinthislabcamefrom.Allthe
plasmids,buffers,enzymes,ladders,etc.wereboughtfromNewEnglandBiolabs(abbreviatedas

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NEB).(Scott2014,pers.comm)Theplasmidcode#Ireceivedwas1236C38andlikeeveryoneelse,
wehadnoideawhattheconcentrationofourDNAwas,somostsimplyassumedthatwehadatleasta
concentrationof150.However,itwaslatermentionedtoexpectaconcentrationof250(Scott2014,
pers.comm.)Oncewehadthisvalue,wecalculatedhowmuchminiprepweneededforeachofour
digeststohave.Inourcase,aswewanted500ng(nanograms)ofDNAineachtest.Wewereableto
calculatethatweneededtouse2L(microliters)ofourplasmidDNA.BeforeIbegantowriteoutthe
restofmydigestrecipes,Ihadtoplanoutwhatrestrictionenzymestouse.AsIdidmorethanone
attempt,Iranatotaloffivedifferentenzymes,butfornowIwillonlytalkaboutmyfirstattempt.Formy
firstattempt,IchosetousePstIformysingledigestandacombinationofBgLIandBamHiformy
doubledigest.OnceIhadmyenzymes,Ihadtodiscoverwhichbuffertouse,andamazinglyIfound
thatPstI,BgLII,andBamHiallusedNEBBuffer3.
OnceIhadallofthat,Iwasfinallyabletocreatemydigestrecipes.Thetotalamountwecould
haveineachofourgelwasatmost25L,soforourDNAdigests,Iwrotemyrecipetototalupto20
L.Therecipeforthecontrolneeded2Lofminiprepplasmid,2LofBuffer3,and16LofdH
2
O.
Forthesingledigest(PstI),IneededtheexactsameexceptthatIusedadifferentamountofwaterdue
totheadditionof1LofPstI(15LofdH
2
O).Thedoubledigest(B+B),alsoneeded2Lof
miniprepplasmid,2LofBuffer3butneeded1LofBgLIand1LofBamHiaswellasonly14L
ofdH2O.
OnceIhadpreparedmyDNAdigest,Iincubatedmysamplesforaboutanhourat37C.
Duringthatwaitingtime,IbegantomakemyagarosegelthatIwoulduseforgelelectrophoresis.
RealizingthatIwouldbeexpectedbasepairs(bp)anywherebetween10,0008,000Idecidedto
createa0.8%agarosegel.First,IhadtocalculatehowmuchsolidagaroseIneededtomakea50mL

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gel,whichturnedouttobe0.4g.OnceImeasuredoutthatmuchsolidreagent,Ipoureditintoan
Erlenmeyerflaskandproceededtocreate50mL(milliliters)1XTAEdilutionfromastockof10x.The
10XstockwasmadebycreatingasolutionofTris(12.1grams),GlacialAceticAcid(2.86mL)and
EDTA(0.73grams)andwasbroughttovolumeof250mL.Afterwehadourstock,Icreatedthe
dilutionbyusingavolumetricpipettotake5mLof10xTAEandboughtittomywantedvolumeof50
mL.AfterIhadthedilution,Ipouredintotheflaskandwhenontomicrowavingmysolutionuntilallthe
agarosehadcompletelydissolved.WhentheagarosehadmeltedcompletelyIaddedabout2Lof
ethidiumbromideinordertomaketheDNAglowlateronwhenIfinisheditsgelrun.Andwhenthe
flaskhadcooleddownenough,IpouredmygelintothegeltrayIpreparedearlierandthenwaitedforit
tosolidify(witha10wellcombinserted).
WhilewaitingformygeltosolidifyandmyDNAtofinishdigesting,Iproceededtomakethe
280mL1xTAEIneededtopourintomygelboxformygelrunlateron.Todothis,Iusedthesame
processIusedwhilecreatingthe50mL1XTAEdilution,onlywith28mLbeingbroughtupto280
mL.AfterabitofwaitingandmygelwasreadyandmyDNAdigested,Ibegantopourthe280mLof
1XTAEintothegelbox,makingsuretofilleverywellandthenproceededtoadd4Lof6Xloading
dyeintoeachDNAsample.Finally.onceImixedthecontentsofeachtubetogether,Ibeganloading24
LofeachDNAsampleand5LofNEB1KBladderintotheirappropriatewells.Onceeverything
wasloaded,Iclosedtheboxandranthegelat140volts.IttookaboutanhourfortheDNAtorunand
oncethathadfinishedrunningItookthegeloutofthetray,ItookittotheUVimagingsysteminthe
backoftheclass.IttooksometinkeringandconstantadjustingbutonceIwassatisfiedwiththeclarity
ofmypictureItookapicture.
OnceIhadmypicture,begantotakemyrulerandmeasureoutinmm(millimeters)howfar

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eachDNAbandhadmovedfromthewells.Aftergettingthebasepairvaluesofthe1KBLadder,I
typedthemoutintoExcel,placedthemnexttothebandmigrationdatapointsIpreviouslyfoundand
createdastandardcurve.Withthatstandardcurve,Iwasabletogetanequationthatwouldaccurately
tellmethesizesofmyDNAfragments.Ifeverythingwentaccordingtoplan,Iwouldhavebeenableto
takethosevaluesandcomparethemtotheexpectedvaluesIfoundfromtheNEBcuttertool
(tinyurl.com/NEBcutter)whereIusedacustomvirtualdigestsimulatortogetfragementsizes.Aslong
asIdideverythingcorrectly,IwouldhavebeenabletodeterminewhatplasmidIhad.However,this
wasntthecasesoIproceededtotryeverythingoncemore.
MysecondattemptwasalmostexactlylikeAttempt#1,exceptthatIuseddifferentenzymes
than.Thistime,IranmyplasmidwithPstI,XbaI,andXhoIallassingledigest.Asstatedbefore,PstI
needNEBBuffer3however,XbaIandXhaIneededNEBBuffer4.(Ishouldnotethatduringmy
secondattempt,IusedNEBBuffer3foralltheenzymes,NEBBuffer4wasnotusedatall.Iwill
addressthispointlateron.)Eachofthe3digestrecipesusedinthiscasearesimilartothedigestrecipe
ofthePstIdigestduringAttempt#1,onlyvaryinginwhichrestrictionenzymesused.Thisattempt,I
usedless1XTAEtofillthegelbox,ratherthan280mL,Iused250mL.Thisdilutionwasmadethe
samewayastheotherswiththechangehowmuchstockTAEwasused(25mLinthiscase).Other
thanthosechanges,Attempt#2wasdoneinthesamewayasthefirstattemptwas.Mygelwasmade
theexactsameway,myDNAsamplesweredigestedatthesametemperatureforthesameamountof
time,etc.
Results
AsIvesaidbefore,Iassumedmystartingconcentrationwasabout250.BeforeIdidthis,I

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usedtheNEBcuttersoftware(tinyurl.com/NEBcutter)torunvirtualdigeststofindwhatsizefrgamentsI
couldexpect.ThisiswhatIexpectedtoget:

PstI BgLI&BamHi XBaI XHoI

pAMP 4556 2166,1118,1114,158 Unabletocut Unabletocut
pKAN 3350,923 3155,794,173,88 Unabletocut 4210
pBLU 3940,1316,197 2121,1756,1410,166 5453 Unabletocut

IwouldlaterusetothistabletocomparewithmyownbandmigrationstoseewhichoftheseenzymesI
got.AfterIranmyDNAusinggelelectrophoresis,Itookapictureandthisiswhatmypictureendedup
lookinglike:
Theorderwent
Ladder,Control,PstI,
B+B,andfinallythe
secondLadder.
However,thecontrol
inthisattemptdidnot
showup,butthat
wasntasimportantas
thedataIreceivedfrommystandardcurve.WhenIcreatedastandardcurvethisisthegraphIended

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upmaking:

Fromthisgraphandtheequationgiven,IcoulddeterminethatmydatawasveryaccurateasmyR2
valuewasverycloseto1,meaningthatitwasalmostspoton,onwheremybandshouldvemoved.I
usedtheequationinthetoplefttocreatethistable:
BandMigration(mm) FragmentSize(bp)
"P"Band1 27.1 6784.17
"B+B"Band1 30 5105.88
"B+B"Band2 32.5 3996.39
"B+B"Band3 36.2 2780.95
"B+B"Band4 39.1 2092.99

WhenIcomparedthistabletomyexpectedfragmentsizetable,Inoticedthatthesingledigest

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producedinaccuratedata,(Iwasntsupposedtogetover5500basepairs)soIwasntabletouseit.
Thehighlightedportionoftableweretheonlyreliablepointsofinterests.AccordingtoMr.Scott,its
possiblethatcertainDNAfragmentswerecoiledtogethermakingitseemliketheyare2bandswhenin
reality,itcouldbe4bandsthatgotcuttogether.(Scott2014,pers.comm)Fromthis,itseemedthatthe
onlypossibleoptionwaspBLUhowever,asmyresultswerentasclearasIwouldhavelikedthemto
be,Itriedoncemorewithenzymesspecifictoeachplasmid.
ForAttempt#2,thisisthepictureIgot:
Mywellorderthistimewent:
Ladder,Control,PstI,XbaI,XhoI,
andLadder.Thistimethecontrol
showedupandbyusingthe
control,Iwasinstantlyableto
eliminateXbaIandXhaIasthey
linedupperfectlywiththecontrol,sotherestrictionenzymesmusthavenotcut.Still,myPstIdigest
endedupworkingmuchbetterthistimearoundandIdecidedtogoaheadandmakemygraph.My
standardcurveendeduplookinglikethis:
Ialsocreatedatable:

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BandMigration(mm) FragmentSize(bp)
Control1 26.2 7315.91
"Pst"1 28.8 5819.72
"Xbal"1 26.2 7315.91
"Xhol"1 26.3 7251.81
Onceagain,thehighlightedportionistheonlypointIcanuse,andasitsbasepairsizeismuchmore
thanotherplasmid,IcanonlycometotheconclusionthatmyplasmidispBLU.
Conclusion
Althoughmydataisntverysupportive,Icametotheconclusionthatmyplasmidisindeed
pBLU.EventhoughIcannotusethisdatatosaymyplasmiditpBLU,itallowedtometoeliminatethe
otherpossibilities.AccordingtoAttempt#1sdatatable,mybasepairsizesformydoubledigestwere
2780.95and2092.99,whenIshouldhavereceivedfourDNAcutsfrommydoubledigestbeing:2121,
1756,1410,166.ThismeansthatsomeDNAbandswerecuttogether,whichwouldnthaveallowed
metoseeallfourexpectedbands.IfpBLUwascutonlyonceasitdidinAttempt#2,Icouldhave
anywherefrom+/250basepairsfrom5453basepairsandaccordingtoAttempt#2stable,PstI
gavemebasepairsizesof5819.72.EventhoughbothmyattemptswerefaroffoffromwhatI
expected,itsstilltoofarofffromtheotherexpectedplasmidsizestoconsidermyplasmidwasanything
otherthanpBLU.AnotherreasonIbelievethatthisispBLUisduetootherswhoexperiencedsimilar
problemsasIdid.SomeonedidthesameexactthingIdid,usingthesameenzymesandbuffersand
endedupwithanalmostidenticalpicture,graphandtable.(Huff2012,perscomm).Weboth
experiencedthesameproblemsandbothcametotheconclusionthatourplasmidcouldntbeanything
butpBLUastherecouldbenootherplasmidthatcouldproducesuchlargefragments.Thesamething

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alsohappenedtopeoplewhousedcompletelydifferentenzymesbutourpicturesandgraphslooked,
onceagainalmostidentical.(Johnston2012,perscomm).ItseemedthatallthosewithpBLU
experiencedsimilarproblemsasIdid,givingmeanotherreasontobelievemyplasmidispBLU.
AsforallthethingsthatcouldhavegonewrongandthethingsIwoulddodifferentlythenext
time,theresalot.OnemistakeImadewasduringAttempt#2whenIendedupusingBuffer3rather
thanusingBuffer4.However,accordingtotheNEBBufferwebsite,Buffer3shouldhaveworked
eitherwiththesameorwithslightlylesssuccessasBuffer4.WhenIcomparedthispictureto
someoneswhobelievedwehadthesameplasmidandhadusedthesameexactenzymes(withthe
correctbuffer)ourpicturesandgraphsendeduplookingalmostidentical.(Huff2014,pers.comm).
ThisleadsmetoconcludethattheBufferworkedasitshouldandallowedtheenzymestocut,butthe
plasmiditselfwasproducingproblems.AnotherpossibleerrorsincludenotdigestingtheDNAlong
enough,notmixinginmyenzymesaswell,orevennotaddingDNAtocertainsamples.ThenexttimeI
dothiskindoflabagain,Iwouldmakesuretousethecorrectbufferforeveryenzyme.Icouldalso
digestmyDNAforlongerperiodsoftimeandtriplechecktomakesureIaddedDNAtoeverysample
Iwantedtorunthroughgelelectrophoresis.HopefullyeverythingwillendupworkingifItrytodothese
certainthings.Still,ImgoingtosaymyplasmidispBLU.

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WorksCited
Nature.com.NaturePublishingGroup.Web.01May2014.
<http://www.nature.com/scitable/definition/plasmidplasmids28>.
"GelElectrophoresis|BiotechLearningHub."BiotechnologyLearningHubRSS.Web.01May
2014.<http://www.biotechlearn.org.nz/themes/dna_lab/gel_electrophoresis>.
"RestrictionEnzymes."RestrictionEnzymes.Web.01May2014.
<http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.html>.

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