LynDao

Biotechnology1015PlasmidIdentification

Introduction:
Biotechnology,atitssimplestdefinition,istechnologicalproceduresbasedonthe
conceptsofbiology.Byutilizingandcapturingtheadvantagesofferedbyorganismsandtheir
products,researcherscanexploitthemforhumanbenefits.Withmoderntechnology,processes
ofexperimentationmaybecarriedouttofiddlewithDNAtoreplicate,splice,orseparateits
fragments.Specificallyspeaking,thecloningofDNArequiresplasmids.Plasmidsarestructures
incellsthathavetheabilitytoreplicateindependentlyonthechromosomestheyaretypically
usedtomanipulategenes.ThesplicingofDNAnecessitatesrestrictionenzymes,whichhavethe
roleofcleavingDNAmoleculesatorneartheprecisesequenceofbases.Whenscanninga
DNAmolecule,therestrictionenzymelooksforaparticularsequence.Oncetherecognition
sequenceisfound,theenzymeproceedstocutthephosphodiesterbackboneofthedouble
strandedDNA.GelelectrophoresisisamethodofsunderingthefragmentsofDNA.Agelof
agaroseiscreatedwith610holes,calledwells,whicharefilledupwithDNAsamples.When
runningtheelectrophoresis,thegelwellsarepositionedclosesttotheblackreceptor(sinceDNA
isnegativelycharged,itwillruntowardspositivered),andiscompletelysubmergedinabuffer
bythenameof1XTAE.TheDNAsamplesarethenloadedintothewells,andvoltagesaresent
intothegelthroughcablesandconnectorstoseparatethesnippetsofDNA.Afterthegelhas
beenallottedtorununtilthebandshavemigratedbetweenthe4and5mark,thegelmaythen
betransferredtoatransilluminatorwhereaphotomaybetakenfordataanalysis.Inthis
experiment,allthreetechniqueswereharnessedtodiscovertheunknownplasmidgiven.

Method:
Inordertodiscovertheplasmid4763A62,3restrictionenzymesweredigestedtogether
withtheminiprepalongwitha3.1bufferanddeionizedwater.Thecombinedsubstancesina1.5
mLmicrocentrifugetubewerethenloadedintoanagarosegel.Theconcentrationoftheplasmid
ineachtubewasdeterminedthroughtheequationx=ngL,andhasbeenconfiguredinthechart
below:

Tube

Miniprep

Buffer
3.1

dH
O
2

BamHI

PvuII

EcoRV

Total
Volume

Single

4l

2.5l

17.5l

1l

25l

Double

4l

2.5l

16.5l

1l

1l

25l

Control

4l

2.5l

18.5l

25l

Assoonastheenzymes,plasmid,buffer,andwaterwereadded,thetubesweremicrocentrifuged
tocombinethesubstances,andplacedonathermalcyclerfor1hourat37tobedigested.The
percentagaroseinthegelexertedinthisexperimentwascalculatedthroughtheequation:1%
gram
/100
mL
=1
gram
/100
mL
=0.5
grams
/50
mL.
Therefore,itwasadamantthat0.5gramsof
agarosebeentailedwith1XTAEtoproducetheneededgel.Asafollowup,5microlitersof
ethidiumbromide(aknownmutagenthatshouldnevercomeintocontactwiththeskin)was
crucialtoexposetheplasmidcuts.Whenthedigestionwascompleted,itwasmixedtogether
witha6xloadingdye.Fromtheequation6x=__l+x,thedeterminedamountofloadingdye
was5l.Beforeanysamplesaretobemicropipettedintothegel,theagarosegelmustbe
completelysubmergedin250280mLof1XTAEbuffer.Ontheoutermostwellsonbothsidesof
thegel,5lof1kbladderwasinserted.24microlitersofthesingle,double,andcontrol(withthe
loadingdyealreadyadded)wereloadedinafter,andthegelwassettorun.Thetransilluminator
systemisappropriatelyaccessibletosnappicturesoftheagarosegeloncetheDNAbandshave
ranbetweenthe4and5mark.Evidently,tocalculatethesizesoftheDNAfragments,aruler
wasusedtoquantifyeachbandwhosenumberswerethenrecordedinGoogleExcelinorderto
computetheequationforthefragmentsizes.

ResultsandConclusions:
Thesingleanddoubledigestbothcorrelatewithaspecificchart
onlinethatwillaidinuncoveringtheunknownplasmid.

pKAN:

DoubleDigest

FragmentSize(kp)

SingleDigest

FragmentSize(kp)

EcoRVPvuII

1951

BamHIBamHI

3287

PvuIIEcoRV

1257

BamHIBamHI

923

PvuIIPvuII

572

EcoRVEcoRV

430


pBLU:

DoubleDigest

FragmentSize(kp)

SingleDigest

FragmentSize(kp)

EcoRVPvuII

1263

BamHIBamHI

3940

PvuIIEcoRV

1013

BamHIBamHI

1316

PvuIIPvuII

726

BamHIBamHI

197

PvuIIEcoRV

535

EcoRVPvuII

459

PvuIIPvuII

453

EcoRVEcoRV

425

EcoRVPvuII

306

EcoRVEcoRV

252

PvuIIEcoRV

21

pAMP:

DoubleDigest

FragmentSize(kp)

SingleDigest

FragmentSize(kp)

EcoRVEcoRV

1286

BamHIBamHI

4556

PvuIIEcoRV

1257

EcoRVPvuII

1117

PvuIIPvuII

896


LadderDoubleSingleControlLadder

BandMigration
(mm)

LadderSize(kp)

LadderBand(1)

13.5

10.0

LadderBand(2)

15.0

8.0

LadderBand(3)

16.0

6.0

LadderBand(4)

17.5

5.0

LadderBand(5)

18.5

4.0

LadderBand(6)

20.5

3.0

LadderBand(7)

23.0

2.0

LadderBand(8)

25.0

1.5

LadderBand(9)

29.0

1.0

LadderBand(10)

34.0

0.5

SingleDigestBand
(1)

18.0

4.660766453

DoubleDigestBand
(1)

19.0

4.031666885

DoubleDigestBand
(2)

20.0

3.487481734

DoubleDigestBand
(3)

21.0

3.016749446

DoubleDigestBand
(4)

22.5

2.427058207

DoubleDigestBand
(5)

23.5

2.099459456

Albeitthedoubledigestcouldnotbeaccountedfortowardsidentifyingtheunknownplasmid
duetothefactthatthisparticulardigesthadbeenincorrectlyloaded,whichcausedtheladderto
bleedintothedigest,thebandswerestillmeasuredandrecordedintoGoogleExcel.Thesuccess
ofthesingledigestgavewaytotheconclusionthatpAMPwastheplasmidbecausethesingle
digesthadmadeexactlyonecut,approximatelyaround4500kp.ForpKANandpBLU,the
singledigestwouldhaveneededtomake2to3splices,notjustone,withthefragmentsizes
rangingfrom197to3940kp.Somehumanerrorsthatoccurredwastherewasanuncertaintyof
whetherornottheethidiumbromidehadbeenadded(whichledtothecreationofanewgel),
andthedigestsmayhavelostaccuracyfromthethermalcycler.Ifthisexperimentweretobe
repeated,oneshouldtakecautioninkeepingtrackoftheethidiumbromidebyphysically
markingdownwhenithasbeenaddedtotheagarosegel,andtobesafe,leavethedigestsonthe
thermalcyclerfor2hoursinsteadof1.Conclusively,basedontheconfigureddata,theunknown
plasmid4763A62ispAMP.

Bibliography:
https://www.bio.org/articles/whatbiotechnology
http://www.nature.com/subjects/biotechnology
http://askabiologist.asu.edu/plasmids
http://askabiologist.asu.edu/restrictionenzymes