LeanaTran

IdentificationofanUnknownPlasmid
Introduction
Asingleexperimentwasperformedtoidentifyanunknownplasmid.Aplasmidisacircularand
doublestrandeddeoxyribonucleicacidthatcontainsafewgenes.Plasmidsareusuallyfoundinbacterial
cellsandareusedtoisolateandmultiplyacertaingene.Scientistshaveoperatedplasmidstoclone,
transfer,andmanipulategenes.Restrictionenzymes(fromNewEnglandBiolabs)alsoknownasrestriction
endonucleasesareDNAcuttingenzymesthatcutthedesiredpieceofDNA.Cutsaremadeatthe
specificnucleotidesequence.TocutadifferentDNAsequencesitsrequiredtogetadifferentrestriction
enzymesthatrecognizesthesequence.Withthetechnologicaluseofthegelelectrophoresiscreatedinthe
1970s.ThetooluseselectricitytoseparateDNAfragmentsthroughtheagarosegel,whichisagellike
substancecreatedwithTAEandanagarosemixture.Asthegelsolidify,thephysicalappearanceofthe
gelreceivesafoggywhitecolor.Nextinlineistotakeoutthe10wellcomb.Asthecombistakenout,10
wellsarepresent.Itisreallyimportantthatthewellsarealignedwiththenegativeendofthebox.The
electrophoresisboxwillcontainapositiveandnegativeend.Oncethewellsarefacingtherightdirection,it
isalsoimportantthattheelectrophoresisboxisfilledwithTAEbeforepipetingthesamplesintothewells
with:theladders,single,anddoubledigests.Theladdersattheendofthewellswillhelpdepictthesizeof
yourproduct.Now,itstimetorunthegel!Plugtheelectrodesintotheelectrophoresisapparatus,withred
coordinatingwithredandsoforth.Letthegelrunforaboutanhouruntilthedyemigratesto45cm.The
smallerthemoleculesthequickertheymovethroughthegel.Thegelisthenobservedunderanultraviolet
(UV)imager.Thebandsarepresentandareseenasbrightfluorescentlines.Thebandsarevisibledueto
theethidiumbromidethatwillattachtotheDNA.

Methods/Materials
Thecodeoftheunknownplasmidwas4522085.Accordingtotheagenda,identifyingthe
concentrationwasfirst.Givenbytheinstructor,theconcentrationwas150nanograms/mL.Forthe
restrictionenzyme,nextstepwastodeterminetheamountofminiprep,10XTAEBuffer,anddH20
requiredtoaddtoeach1.5microfugetube.Toidentifytheamountsofeachsolution,usetheconcentration
andmultiplyitwiththevolume(ul)=desiredconcentration.Asshownbelowtheequation,(Your
concentrationng)/(ul)*(x)(ul)=500ng.150ng/500*500ng/500.Theoutcomefromthe
equationis3.3ulofDNAplasmid.Tofindtheamountof10XTAEBuffer3,thefollowingmathand
equationisnecessary,(10x)(vl)/10=(1x)(20ul)/10=2ul.Throughthemath,wewouldneed2ulof
10XTAEBuffer3inthisexperiment.Inthedatatablebelowitexplainshowmuchbuffer,dH20,and
minipreptoadd.Inthesingledigest,IchosetodoPvul.ForthedoubledigestIchosetodoNdel+Xhol.
Inthesingledigest,youwouldadd3.33ulofminiprep,2ulof10XTAEBuffer3,1ulofPvul.and13.67
ulofDH20leadingtothetotalvolumeof20ul.TheDH20willbringthecontenttovolume.Inthecontrol,
therewillbenoenzymesusedfromthesingleanddoubledigest,itwouldonlyconsistofMiniprep,10X
Buffer3,andDH20.Featuredinthedoubledigestwouldbe3.33ulofminiprep,2ul10xTAEBuffer3,
12.67ulDH20,1ulofNdel,and1ulofXhol.Eachmicrofugetubeshouldhold20ulofsolutions.

Tubes Miniprep
(ul)
10X
Buffer3
Dh20(ul) Pvul Ndel XHol Total
Volume
(ul)
Control 3.33 2ul 14.67 20
Pvul
(Single)
3.33 2ul 13.67 1ul 20
Ndel+
Xhol
(Double)
3.33 2ul 12.67 1ul 1ul 20

Afteralltheliquidsareaddedtoeach1.5microfugetubeaccordingly,thethreetubeswillbeincubatedat
37.0Cforonehour.Whilethetubesareincubatinginthewaterbath.Itwastimetopreparetheagarose
gelforthegelelectrophoresis.First,Ineededtofindthepercentofagarosegelneeded.Iwasabletofind
thepercentneededwiththemath:
60mL*8/100mL=.48g
Afterthemeasurement,Ipouredtheagarosesolidpowderreagentintoa250mLErlenmeyerFlask.
Afterward,Ineededa1XTAEbuffer,thatrequiredmetomakeadilution.ThroughthemathbelowIwas
abletointerprettheamountofmlneeded.
(60mL)(1x)/10=(?mL)(10x)/10
=6mL
Iplaced6mLofthe10XTAEBufferthenbroughtittovolumewithdH20to60mL.Thesolutionwas
pouredintoflaskwithagarose45secondswith(15secondsincrements).Afterward,asthesolution
becomestransparent,thenyouwouldaddthe1ulofethidiumbromide.TheEthidiumBromidewillattach
itselfwiththeDNAmakingitglowintheUVpicturetakenlater.Oncetheethidumbromideisaddedand
thesolutioniscooledenough,pourthesolutionintotheelectrophoresistray.Toensurethesolutiondoesn't
leakaroundthebox,makesurethegeltrayisturnedtothepointwheretherubbergasketsaretouching
againstthewallsofthegelbox.Oncethetrayissturdy,pourtheagarosesolutionintothegeltray.
Afterward,addthegelcombthatcreates10wells.Letthegelsolidifyforabout45minutestoanhour.
WhilethegelissolidifyingitwastimetocreatetheTAEbuffersolutiontosubmergethegelinto.What
wasneededwasa280mlof1XTAE.Icreateda10Xbuffer,hadtomakeanotherdilution.Tocreatethe
dilutionIfollowedthemath/equationbelow.
280mL8(1x)/10=10x*(?)/10
Afterfiguringoutthemath,Ineeded28mLof10XTAEbufferandbroughtthatuptovolumewithDH20
to280mL.Asthegelsolidified,itwastimetotakeoutthe10gelcombandalsoturnthetraysothewells
areparalleltotheblack(negativeside)runningtored(positiveside).AfterIturnedthegeltray,Ipoured
thedilutedTAEbufferintothegelboxsubmergingthegel.Nextontheagendaistopippettheloadingdye
intoDNAsolution.Iplaced6ulof6xloadingdyetoeachmicrofugetube.
(5X)(Xul)=(1X)(30ul)
5*?=30?=6
Aftertheloadingdyewasplacedinwiththethreemicrofugetubes,Istartedoutbyplacingtheladdersin.
Iloaded5ulof1KbNEBladderinthewell.OnceIstartedthewellswiththeladder,Iloaded24ulofmy
samplessimilartothefigurebelow.

Ladder Control Pvul Ndel+

Xhol
Ladder

OnceIloadedthesamplesintothewells,Iplacedthelidontheelectrophoresisboxandranitat140volts
foronehour.AstheDNAisrunningonceitisbetweenthe45centimetermark,Itookoffthegeland
turnedofftheelectrophoresisbox.ItooktheremovedthegelfromtheboxandtookaUVpictureofthe
gel.WiththecorrectadjustmentsonthecameraItookthepictureofthegel.TheUltravioletimagerwas
abletogivememyresults.OnemistakeIdidwasnotzoomintomypicture.Afterprintingouttheimage,I
wasabletomeasureeverybandandmakeatableofthedistancesthesamplestraveled(mm).TheDNA
codeswereonTinyurl.com/plasmidseq.IwasabletouseTinyurl.com/NEBcuttertofindthefragment
sizes.

Conclusion
Thebestfitoflineequationisy=71881e^0.126xandR2=0.9948.Throughthedata
collecteditisbelievedthatitspAMP.Thebasepairsarecloselysimilartothedatacollected.Factorsthat
couldaffectmydataisthetimemyseconddigestwasincubating.Itwouldbeunderdigested.Toidentify
myplasmidaspAMP,causeIcomparedthecustomdigestfromNebcutter.Pvulis3643BPfromNeb
cutterandthestandardcurveisat3919.42,thatwastheclosestBP.

Sources:
http://www.nature.com/scitable/definition/plasmidplasmids28
http://www.dnalc.org/resources/animations/gelelectrophoresis.html
http://eebweb.arizona.edu/blast/Gel%20electrophoresis.html
Tinyurl.com/plasmidseq
Tinyurl.com/NEBcutter