ElaineEm

PlasmidIdentificationFinal

Introduction:AplasmidisacircularpieceofDNAthatexistsapartfromthechromosomeanditcan
replicateindependently.Theycanbefoundinbacteriawithmultiplereasonsbehindit.Plasmidsare
mainlyusedinlabsforthemanipulationofgenes.Theplasmidscancarrygenesthatmakethemresistant
toantibiotics.TheplasmidscanbeusedtoinvestigateRNAsandpromoters.Restrictionenzymesare
enzymesthatonlycutDNAinveryspecificrecognitionsequences.Restrictionenzymesarecrucialto
recombinantDNAtechnologywithoutrestrictionenzymes,scientistswouldntbeabletocuttheDNA
toacertainlengthandallowfortheattachmentofnewDNA.Therestrictionenzymescouldalso
measurehowlongtheDNAstrandis.GelelectrophoresisisusedtoseparatemixturesofRNA,DNA,
andproteinsaccordingtotheirmolecularsize.GelelectrophoresisbeginswithamixtureofTAEasmall
amountofagarosetomakeanagarosegel.Witha10wellcombinsertedintothegelbeforeitsolidifies,
itcreateswellsforthesolutionstobeaddedto.TheDNAandDNAladderswouldbeinjectedintothe
wellsforgelelectrophoresistotakeplace.Themoleculesfromthesolutionsareseparatedastheyre
beingpushedbyanelectricalforcethroughsmallfiltersintheagarosegel.Thelengthsofhowfarthey
travelandtheirsizescorrespondwitheachother.Thetimeforthegeltorunwillusuallytakeuptoan
houratthemost.ThegelwouldberemovedandmovedtotheUVboxinordertotakeapicturewith
UVlightsshiningontothegel,afterthepictureistakenitwouldbepossibletodeterminethedistanceof
theDNAbymeasuringfromtheladderbasepairlengthstofindthedistanceoftheunknownDNA.The
goaloftheexperimentistodeterminetheunknownplasmidthatweweregiveninthebeginningofthe
experiment.Findingtheunknownplasmidmaybeimportanttomyunderstandingofbiotechnologylab
techniquestogetanunderstandingofhowtouserestrictionenzymes,micropipettes,gelelectrophoresis,
andoveralleverythingIhadlearnedinmybiotechnologylabtoputtouseinthefinalexperiment.The
strategyIhadusedtoachievethisgoalwouldincludetheuseofrestrictionenzymestocuttheplasmidin
auniquewayinordertodeterminetheunknownplasmid(s)(pKAN,pBLU,pAMP)withpreciseand
cautiousmeasurementsthroughoutthelab.
Methods:Theconcentrationwas150nanograms/microliter.InordertofigureouthowmuchDNAis
neededthefollowingequationwouldbeused,(plasmidconcentration*volume(ul)=desired
concentration).Thedesiredconcentrationhadturnedouttobe500ng/ulfortheexperiment.Another
equation,150ng/ul*v(ul)=500ng/ulisusedandresultsto3.3uloftheDNAplasmid.1ulof
restrictionenzymeisalsoaddedtotheDNAplasmid.InmysingledigestIhadadded1ulofPstIwhich
wascreatedbyNewEnglandBiolabs(NEB).InmydoubledigestIhadincluded1ulofrestriction
enzymeHindIIIandrestrictionenzymeNdel,whichwerebothcreatedbyNEB.Thetotalvolumeof
eachmicrotubewouldaddupto20ul.1xbufferwouldbenecessarytoaddtothesolution.Formy
singledigestcontainingrestrictionenzymePstIIaddeda3.1NEBuffer.Tomydoubledigestcontaining
restrictionenzymesofbothNdelandHindIIIIwouldadda2.1NEBuffertoit.Afteraddingtherestof
therestrictionenzymesIwouldbringittovolumebyaddingaspecificamountofdH2Ointoeachtube
(asseeninthetablebelow).

Typeof
Digest
Plasmid
DNA
2.1/3.1
NEBuffer
PstI Ndel HindIII dH2O Total
Volume
Control 3.3ul 2ul 14.7ul 20ul
Single 3.3ul 2ul 1ul 13.7ul 20ul
Double 3.3ul 2ul 1ul 1ul 12.7ul 20ul

AfterincludingallofthenecessaryproductsintothemicrotubesIwouldincubatethemat37.0
Cfor2hours.WhilethemicrotubeswereincubatingIhadcreatedthegelforthegelelectrophoresis.I
hadgatheredaspecificamountof.8%ofagarosegelthatwouldbeabletomeasurefrom80012,000
basepairs.Agarosegel,TAEbuffer,and1ulofethidiumbromideismixedtogetherasasolutionfora
50mlgel.Theequation.8g/100ml=x/50mlwasusedtofindxwhichwouldbetheamountofthe
agaroseneeded.Theamountofagarosethatwasdeterminedfromtheequationwas.4grams.The
agaroseisaddedintoaflaskwith10xTAEbuffer.Theamountof10xTAEbufferthatwasneededwas
determinedthroughanequationof50ml*1X=10X*x,lookingforx.5mlofthe10xTAEbufferwas
addedintotheflaskandbroughttovolumeof50ml.OnceIhadincludedalloftheproductstogetherI
wouldswirltheflaskbeforeputtingintothemicrowavefor50secondsuntiltheagarosemixwasnt
visibleanymore.Later,1ulofethidiumbromideisaddedintotheflaskwiththeagarosemixtureasit
coolsdowntobeabletopourintothegeltray.AfterpouringinthesolutionintothegeltrayIwould
adda10wellcombintothetraybeforeithardens.Thegelwouldhardenwithinhalfanhour,while
waitingforittohardenIhadbegantomakeaTAEbuffertocovertheentiregelduringthegel
electrophoresis.Iwouldadd280mlof1xTAEbufferstartingoutwitha10xTAEbuffer.Theamount
ofTAEbufferneededwouldbedeterminedbytheequation280ml*1XTAEbuffer=10XTAE
buffer*x,insearchofx.Aftersolvingtheequation,28.mlofthe10XTAEbufferwouldbebrought
tovolumeto280ml.WhenIknewthegelhadhardenedIremovedthegelfromthegeltraywiththe
combandturneditsothatthewellswouldbeclosertotheblack()andacrossthered(+).The280ml
ofbufferIhadjustmadewouldbeaddedontopofthegelintothegeltray.4ulofloadingdyewould
beaddedtoeachoftheDNAtubesaftertheincubationwouldbeoverwiththeequationof,20ul*1X
DNA=6Xdye*x.AfterincludingtheloadingdyesintoeachofthemicrotubesIwouldinject25ulofthe
DNAintothewells,withthatIwouldadd5ulofthe1kbladder(NEBmade)Withthetable/pattern
belowishowthewellsofthegelwasloaded.

ladder control single double ladder

Lastly,Isetthegeltorunat140voltsforabout45minutes.OncetheDNAreachedbetween
the45centimetersmark,IremovedthegelfromtotakeapictureoftheDNAbandswiththeUVlight.
OnceIhadprintedtheimageIhadtookfromthemachine,Iwouldcreateareferencepointand
measurefromthebottomofeachbandinmillimeters.AfterfindingthedistancesIwouldcreatea
standardcurvewhichwas,y=15464e^.123x,IhadusedMicrosoftExcelinordertofindmystandard
curve.IwasabletofigureoutmyfragmentsizebyusingtheDNAcodesfrom
tinyurl.com/plasmidseq.Iwouldthenfindmyfragmentsizebyusingtinyurl.com/NEBcutter.Two
otherwebsiteswereusedsuchastinyurl.com/nebbuffersinordertofindthebuffersthatareusedina
relationshipwiththeenzymes.tinyurl.com/nebdoubledigestwasalsousedtofindouthowenzymes
andbuffersworktogether.

ResultsandConclusion:BelowistheStandardCurveIhadcreatedusingMicrosoftExcel.

HereismyGelElectrophoresispicturewiththelinesdrawnonittofigureoutthefragments.

PstI,singledigestsizes NdelandHindIII,Double
DigestSize
pAMP 4539 2118,2421
pKAN 923&3271 2096,2098
pBLU 197,1316,3924 3006,2431
UnknownPlasmid

InthisexperimentIhaveconcludedthatIhavepKANwithaconcentrationof150ng/ml.There
weremanythingsthatwentwiththis.pKANweretheclosestnumberstomycuts.

References
http://tools.neb.com/NEBcutter2/
http://www.dnalc.org/resources/plasmids.html
https://www.neb.com/toolsandresources/interactivetools/doubledigestfinder