All plant biotechnology related experiments such protoplast isolation, plant genome etc.

1. 1
2017
ABHISHEKKAUSHIK
01-Jul-17
PLANTBIOTECHNOLOGYEXPERIMENTS
4thSem

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Abhishekkaushik
1.AIM: To prepare Murashige and Skoog (MS)
mediaforplanttissueculture.
PRINCIPLE:
Thebasalmediumisformulatedsothatitprovidesallofthe
compoundsneededforplantgrowthanddevelopment,
includingcertaincompoundsthatcanbemadebyanintact
plant,butnotbyanisolatedpieceofplanttissue.Thetissue
culturemediumconsistsof95%water,macro-and
micronutrients,vitamins,aminoacids,sugars.Thenutrients
inthemediaareusedbytheplantcellsasbuildingblocksfor
thesynthesisoforganicmolecules,orascatalysatorsin
enzymaticreactions.Themacronutrientsarerequiredin
millimolar(mM)quantitieswhilemicronutrientsareneeded
inmuchlower(micromolar,μM)concentrations.Vitamins
areorganicsubstancesthatarepartsofenzymesor
cofactorsforessentialmetabolicfunctions.Sugaris
essentialforinvitrogrowthanddevelopmentasmostplant
culturesareunabletophotosynthesizeeffectivelyfora
varietyofreasons.Murashige&Skoog(1962)medium(MS)
isthemostsuitableandcommonlyusedbasictissueculture
mediumforplantregeneration.
MATERIALSANDREQUIREMENTS:
· Distilledwater,sucrose,gellingagent,1M NaOHor1M HCl
toadjustthepH,micronutrientsstock,macronutrientsstock,
organiccompoundstock.
· Equipment–Balance,pHmeter,autoclave,LAFcabinet.
· Glassware–conicalflask,phytajars,beakers,pipettes.
Preprationofmacronutrientstock
MSmajorsalts mg/1Lmedium 500mlstock(20X)
1.NH4
NO3
1650mg 16.5gm
2.KNO3
1900mg 19gm
3.Cacl2
.2H2
O 440mg 4.4gm
4.MgSO4
.7H2
O 370mg 3.7gm
5.KH2
PO4
170mg 1.7gm
Preprationofmicronutrientstock
MSminorsalts mg/1Lmedium 500ml
stock
(200X)
1.H3
BO3
6.2mg 620mg
2.MnSO4
.4H2
O 22.3mg 2230mg
3.ZnSO4.
4H2
O 8.6mg 860mg
4.KI 0.83mg 83mg
5.Na2
MoO4.
2H2
O 0.25mg 25mg
6.CoCl2.
6H2
O 0.025mg 2.5mg
7.CuSO4.
5H2
O 0.025mg 2.5mg
Preprationofvitaminstock
MSVitamins mg/1Lmedium 500mlstock
(200X)
1.Thiamine(HCl) 0.1mg 10mg
2.Niacine 0.5mg 50mg
3.Glycine 2.0mg 200mg
4.Pyrodoxine(HCl) 0.5mg 50mg
PREPARATIONOFHORMONESTOCKS:
· Weighout0.3gofIAA
· SterilizetheIAAbyplacingunderUVlightfor30mins.
Transfertoasterilevolumetricflask.
· DissolvetheIAAinfewdropsofethanolandmakeupthe
volumeto100mlusingsteriledistilledwater.
· Weighout0.1mgofkinetinandsterilizeunderUVlight
· Transferto a flaskand dissolvein 100 mlofsterile
distilledwater
· Add30mlofIAAstockand1.5mlofkinetinstocktoeach
bottleaseptically.Mixwellandallowthecontentstosolidify
anduseimmediatelyorstoreat4
0
C.
PROCEDURE:
1. Dissolve the microelements,macroelements and
organicsin800mlofdistilledwater.
2. Addtherequiredhormonesolutions.
3. Adjustthemedium pHto5.7byadding1M NaOHor
1MHCl.
4. Add additionaldistilled waterto adjustthefinal
volumeto1litre.
5. Addtheagarandheatthemedium todissolvethe
agar.
6. Dispensethemedium intoculturetubesorvessels
andautoclavefor20minutesat121°C.
2.Aim: isolation ofprotoplastby mechanical
method
Principle:
Protoplastcanbeisolatedfrom almostallplantparts:roots,
leaves,fruits,tuber,
rootnodules,pollenmothercelletc.Protoplastisolatedby
mechanicalisacrudeand
tediousprocedure.
Cellsareplasmolysedcausingtheprotoplasttoshrinkfrom
thecellwall.The
protoplastobtainedfrom thismethodisthenculturedon
suitableculturemedium.The
principledeficiencyofthisapproachisthattheprotoplast

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Abhishekkaushik
releasedisfewinnumber.
Mechanicalisolationwasthatofonlyhistoricaleventnow.
MaterialsRequired:
1.Plantleaves–Durantarepens
2.Mortarandpestle
3.PhosphatebufferpH-7.0
4.0.3Msorbitol
5.0.3Mmannitol
6.Glassslides
7.Microscope.
Procedure:
1.Youngleaveswereobtainedfrom plantsgrowingoutdoors
andinitiallywashed
withtapwatertoremoveanydustparticles.
2.The leaves were washed with phosphate bufferand
homogenizedgentlywiththe
mortarandpestle.
3.Thecrudeprotoplastsuspensionwascentrifugedatvery
low50-100rpmfor10
minutes.
4.Thesupernatantcontainingintactprotoplastwascarefully
pipettedoutandthe
pelletcontainingcelldebrisandothercellorganelleswere
discarded.
5.Smallvolumeofsupernatantwasplacedintheslidesand
coveredwithcoverslip.
6.Theslidewasobservedinlightmicroscopetofindout
viableprotoplast
Result:
Thesphericalshapedprotoplastswereobservedusingthe
microscope.
3.AIM: isolation ofprotoplasts by enzymatic
method
PRINCIPLE:
Protoplastsareisolatedbytreatingtissueswithamixtureof
cellwalldegrading
enzymeinsolution,whichcontainosmoticstabilizer.Amost
suitablesourceof
protoplastsismesophylltissuefrom fullyexpandedleavesof
youngplantsornewshoots.
Thereleaseofprotoplastisverymuchdependentonthe
natureandcompositionof
enzymesusedtodigestthecellwall.Therearethreeprimary
componentsofthecellwall
whichhavebeenidentifiedascellulose,hemicellulaseand
pectinsubstance.Pectinase
(macrozyme)mainly degrades the middle lamella while
celluloseandhemicellulase
degradesthecelluloseandhemicellulosiccomponentsofthe
cellwall.Duringthis
enzymatic treatment,the protoplastobtained should be
stabilizedbecausethemechanical
barrierofthecellwallwhichofferedsupporthasbeenbroken.
Forthisreasonan
osmoticm is added which prevents the protoplastfrom
bursting.
MATERIALSREQUIRED:
1.Youngleaves
2.70%ethanol
3.2%cellulose
4.13%mannitol
5.0.5%macrozyme
6.CPWsaltsolution:
KH2PO4-27.2mg/l
KNO3-101mg/l
CaCl2-1480mg/l
MgSo4-246mg/l
KI-0.16mg/l
CaSo4-0,026mg/l
pH-5.8.
PROCEDURE:
1.Theyoungleaveswerecollectedandwashedinsterile
distilledwaterthrice.
2.Theleaveswerecutintosmallbits.
3.Thentheleaveswerekeptimmersedin13%mannitolfor1
hforpre-plasmolysis.
4.Mannitolwas removed afterincubation antsterilized
enzymemixture(Cellulase+
macerozyme)wasaddedandincubatedat25ºCinashaker
for12h
5.Thefiltratewascentrifugedat100gfor5mintosediment
theprotoplast.
6.Thesupernatantwasremovedandtheprotoplastpellet
wassuspendedin10mlof
CPW+21%sucrosesolution.
7.Themixturewascentrifugedat100gfor5min.Theviable

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protoplastwillfloatto
thesurfaceofthesucrosesolution.
8.The supernatantwas collected and viewed under
microscope.
9.Theprotoplastswerevisualizedinmicroscope.
RESULT:
Protoplastswereisolatedbyenzymaticmethodandviewed
underthemicroscope
4.AIM:Thepolymerasechainreaction(PCR)isa
laboratorytechniqueforDNA
Principle
ThePCRinvolvesthe primermediatedenzymatic
amplification ofDNA.PCRisbasedonusingtheability
ofDNApolymerasetosynthesizenewstrandofDNA
complementarytotheofferedtemplatestrand.Primer
isneededbecauseDNApolymerasecanadda
nucleotideonlyontoapreexisting3′-OHgrouptoadd
thefirstnucleotide.DNApolymerasethenelongateits
3endbyaddingmorenucleotidestogeneratean
extendedregionofdoublestrandedDNA.
Materials
BuffersandSolutions
10xAmplificationbuffer
Chloroform
dNTPsolution(20mM)containingallfourdNTPs(pH8.0)
EnzymesandBuffers
ThermostableDNApolymerase
NucleicAcidsandOligonucleotides
Forwardprimer(20μM)inH2O
Reverseprimer(20μM)inH2O
TemplateDNA.
DissolvetemplateDNAin10mMTris-Cl(pH7.6)containinga
low concentration ofEDTA (<0.1 mM)atthe following
concentrations:mammaliangenomicDNA,100μg/ml;yeast
genomicDNA,1μg/ml;bacterialgenomicDNA,0.1μg/ml;
andplasmidDNA,1-5ng/ml.
Method
1. Inasterile0.5-mlmicrofugetube,mixinthefollowing
order:
REAGENTS AMOUNT(μl)
Deionizedwater 37.5μl
Taqassaybuffer(10x) 5μl
TemplateDNA 1μl
dNTPsmix 2μl
Forwardprimer 2μl
Reverseprimer 2μl
TaqDNApolymerase 5μl
Thetablebelow providesstandardreactionconditionsfor
PCR.Mg2+ (1.5 mM) ;KCl(50 mM) ;dNTPs (200 μM)
;Primers(1μM);DNApolymerase(1-5units);TemplateDNA(1
pgto1μg).
2. Ifthethermalcyclerisnotfittedwithaheatedlid,overlay
thereactionmixtureswith1drop(approx.50μl)oflight
mineraloil.Alternatively,placeabeadofwaxintothe
tubeifusingahotstartprotocol.Placethetubesorthe
microtiterplateinthethermalcycler.
3.Amplify the nucleic acids using the denaturation,
annealing,and polymerization times and temperatures
listedbelow.
4. Withdraw a sample (5-10 μl)from the testreaction
mixtureandthefourcontrolreactions,analyzethem by
electrophoresisthroughanagarosegel,andstainthegel
withethidiumbromideorSYBRGoldtovisualizetheDNA.
5. Ifmineraloilwasusedtooverlaythereaction(Step2),
removetheoilfrom thesamplebyextractionwith150μl
ofchloroform.Theaqueousphase,whichcontainsthe
amplifiedDNA,willform amicellenearthemeniscus.
Themicellecanbetransferredtoafreshtubewithan
automaticmicropipette.
AmplificationBuffer:
500mMKCl.
100mMTris-Cl(pH8.3atroomtemperature).
15mMMgCl2.
Autoclavethe10xbufferfor10minutesat15psi(1.05
kg/cm
2
)onliquidcycle.Dividethesterilebufferintoaliquots
andstorethemat-20
o
C.
KCl
DissolveanappropriateamountofsolidKClinH2O,autoclave
for20minutesonliquidcycleandstoreatroom temperature.
Ideally,this4Msolutionshouldbedividedintosmall(approx.
100μl)aliquotsinsteriletubesandeachaliquotthereafter
usedonetime.
Tris-Cl
Dissolve121.1gofTrisbasein800mlofH2O.AdjustthepH
tothedesiredvaluebyaddingconcentratedHCl.
Base wavelength(nm) ExtinctionCoefficient(E)(M-1cm-1)
A 259 1.54x10
4
G 253 1.37x10
4
C 271 9.10x10
3
T 267 9.60x10
3

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Abhishekkaushik
pHHCl
7.470ml
7.660ml
8.042ml
(1M)Allowthesolutiontocooltoroom temperaturebefore
makingfinaladjustmentstothepH.Adjustthevolumeofthe
solution to 1 litre with H2O.Dispense into aliquots and
sterilizebyautoclaving.Ifthe1M solutionhasayellowcolor,
discarditandobtainTrisofbetterquality.ThepH ofTris
solutionsistemperature-dependentanddecreasesapprox.
0.03pH unitsforeach1oC increaseintemperature.For
example,a0.05M solutionhaspHvaluesof9.5,8.9,and8.6
at5
o
C,25
o
C,and37
o
C,respectively.
dNTPSolution
DissolveeachdNTP(deoxyribonucleosidetriphosphates)in
H2Oatanapproximateconcentrationof100mM.Use0.05M
TrisbaseandamicropipettetoadjustthepHofeachofthe
solutionsto7.0(usepH papertocheckthepH).Dilutean
aliquotoftheneutralizeddNTPappropriately,andreadthe
opticaldensityatthewavelengthsgiveninthetablebelow.
CalculatetheactualconcentrationofeachdNTP.Dilutethe
solutionswithH2Otoafinalconcentrationof50mM dNTP.
Store each separately at 70
o
C in smallaliquots.For
polymerasechainreactions(PCRs),adjustthedNTPsolution
topH8.0with2NNaOH.Commerciallyavailablesolutionsof
PCR-gradedNTPsrequirenoadjustment.
Foracuvettewithapathlengthof1cm,absorbance=EM.
100mM stocksolutionsofeachdNTP arecommercially
available.
Precautions
chloroformCHCl3 isirritatingtotheskin,eyes,mucous
membranes,andrespiratorytract.Itisacarcinogenandmay
damagetheliverandkidneys.Itisalsovolatile. Avoid
breathingthevapours. Wearappropriateglovesandsafety
glasses.Alwayswearachemicalfumehood.
5.AIM:Toisolateandinoculateanthersforhaploid
production.
PRINCIPLE:
Haploidsrefertothoseplantswhichpossessa
gametophyticnumberof
chromosomesintheirsporophytes.Haploidsmaybe
groupedintotwobroad
categories:
(a)monoploidswhichpossesshalfthenumberof
chromosomesfromadiploid
species.
(b)Polyhaploidswhichpossesshalfthenumberof
chromosomesfromapolyploidy
species.
Haploidproductionthroughantherculturehasbeenreferred
toasandrogenesis
whilegynogenesisistheproductionofhaploidplantsfrom
ovaryorovuleculture
wherethefemalegameteorgametophyteistriggeredto
sporophyticdevelopment.
MATERIALSREQUIRED:-
1.AnthersfromHibiscus
2.MSmedium
3.growthfactors
4.70%ethanol
5.2%mercuricchloride
6.Mesoinositol
7.Scissors
8.Scalples
9.Petriplates
10.Forceps.
PROCEDURE:
1.FlowerbudsofHibiscuswerecollected.
2.Theflowerbudsaresurfacesterilizedbyimmersingin
70%ethanolfor60sec
followedbyimmersingin2%sodiumhypochloridesolution
for1minorin
mercuricchloride.
3.Thebudswerewashedfourorfivetimeswithsterile
distilledwater.
4.ThebudsweretransferredtoasterilePetridish.
5.Thebudsweresplitopenusingabladeandtheanthers
wereremovedwithout
damageandthefilamentswereremoved.
6.TheantherswereplacedhorizontallyontheMSmedium
supplementedwith
differentconcentrationofplantgrowthregulatorsor
mesoinositol.
7.ThePetriplatesweresealedandincubatedindarkat28ºC.
8.ThePetriplateswereexaminedforthegerminationof
anthers.
RESULT:
Theantherunderwentgerminationleadingtotheformation
ofhaploidplantlets.
6.AIM-RandomamplifiedpolymorphicDNA(RAPD)
PCRbasedtechniqueforidentifyinggenetic
variation.
Principle

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Abhishekkaushik
The RAPD technique is based on the polymerase chain
reaction (PCR).A targetDNA sequence is exponentially
amplifiedwiththehelpofarbitraryprimers,athermostable
DNA polymerase,dideoxy nucleotide tri- phosphates,
magnesium and reaction buffer.The reaction involves
repeatedcycles,eachconsistingofadenaturation,aprimer
annealingandanelongationstep(fig1).Inthefirststepthe
DNAismadesinglestrandedbyraisingthetemperatureto
94C (denaturation).In thesecond step,lowering ofthe
temperaturetoabout40to65Cresultsinannealingofthe
primerto theirtargetsequences on the template DNA
(annealingstep).Inthethirdcycle,temperatureischosen
wheretheactivityofthethermostableTaqDNApolymeraseis
optimal,i.e.,usually720C.
Materialandreagents
Instruments:
PCR machine(PerkinElmer9600),microcentrifuge,100V
powersupply,Gelelectrophoresistank,gelmouldandslot
former,UVtransilluminator,Camera,autopipettes,vortex.
Reagents:
1.TaqDNApolymerase
2.GenomicDNA(5ng/l)
3.dNTPmix(2mMeachofdATP,
dCTP,dGTPanddTTP)
4.MgCl2(25mM)
5.BufferforDNApolymerase
6.10-meroligonuleotideprimers(5M)
7.Steriledistilledwater
8.Electrophoresisgradeagarose
9.0.5XTBEbuffer
10.Ethidiumbromidesolution(10mg/mll)
11.DNAlengthmarker
12.Loadingbuffer
Miscellaneous:ThinwalledPCRtubes,tips,tissuepaper.
Protocol
1.Each10lofreactionmixcontains
Component Volume FinalConcentration
GenomicDNA 3.0l 15ng
Buffer 1.0l 1X
dNTPs 1.0l 0.2mM
Primer 0.6l 0.6M
TaqDNApol 0.2l 1unit
MgCl2 1.0l 2.5mM
Water to10l
Prepareamastermix(forallsamples+control)that
containsalltheabovecomponentsexcepttheDNA.
Thawallcomponentscompletely
VortextheMgCl2vigorously
Vortexthemastermixtomixallcomponentsbefore
aliquoting
2.AliquotintoPCRtubesandaddthetemplateDNA.Mix
well.
3.PlacethePCRplatecarryingthereactiontubesinthe
sampleblockofthethermocycler.
4.Carryoutaninitialdenaturationstepat94Cfor4min
followedby40cycleswiththefollowingcycleparameters:
Step194Cfor1min
Step235Cfor1min
Step372Cfor2min
Extendthe72Cstepofthefinalcycleby5min
5.Whentheamplificationhasfinished,add3loftheloading
dyetoeachsample.
6.Preparea1.2%agarosegelin0.5XTBEbuffercontaining
ethidiumbromide(5g/mlofgel).LoadtheDNAlength
markerandthesamples.Runthegelin0.5xTBEbufferat
55Vfor4h.
RESULTS
VisualizethegelonaUVtransilluminator.Ifrequiredthegel
canbephotographedusingPolaroid665or667filmand
analysedfurther.
7Aim:TheprocessofDNAfingerprintingby
RestrictionFragmentLengthPolymorphism(RFLP)
method.
Principle:
Restrictionfragmentlengthpolymorphism(RFLP)analysisis
extensivelyusedinmolecularbiologyfordetectingvariation
attheDNAsequencelevel.RFLPfunctionsasamolecular
markerasitisspecifictoasingleclone/restrictionenzyme
combination.MostRFLPmarkersarecodominantandhighly
locus-specific.In molecularbiology,restriction fragment
lengthpolymorphism,orRFLPisatechniquethatexploits
variations in homologous DNA sequences.RFLP is a
difference in homologous DNA sequences thatcan be
detectedbythepresenceoffragmentsofdifferentlengths
afterdigestionoftheDNAsamplesinquestionwithspecific
restrictionendonucleases.Thebasictechniquefordetecting
RFLPsinvolvesfragmentingasampleofDNAbyarestriction
enzyme,whichcanrecognizeanddigestDNAwherevera
specificshortsequenceoccurs,in aprocessknown as
restrictiondigestion.TheresultingDNAfragmentsarethen
separatedbylengththroughaprocessknownas agarose
gel electrophoresis. RFLP is specific to a single
clone/restrictionenzymecombinationanditoccurswhen
thelengthofadetectedfragmentvariesbetweenindividuals.
Materials
DNAsample –15.0μl
10XAssayBuffer –3.0μl
MilliQwater* –10.0μl
restrictionenzymes(EcoRIandPstI)
Glasswares:Measuringcylinder,Beaker
Reagents:Ethidiumbromide(10mg/ml)
Other requirements: Electrophoresis, apparatus,UV
Transilluminator,WaterBath,Micropipettes,Tips,
Adhesivetape,Crushedice,Microwave/Hotplate/Burner
Procedure:
1.Beforestartingtheexperiment,crushiceandplacethe
vialscontainingDNAsamples,
restrictionenzymesandassaybuffersontoit.
2.InthisexperimentthreereferenceDNAsamplesandthe
testsamplearedigested
simultaneouslywithtworestrictionenzymesEcoRIandPstI.

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Abhishekkaushik
3.Setupfourseparatereactionmixturesasfollows:
DNAsample –15.0μl
10XAssayBuffer –3.0μl
MilliQwater* –10.0μl
EcoRI –1.0μl
PstI –1.0μl
Total30μl
4.Afterpreparingthefourreactiontubes,mixthe
componentsbygentlepipettingandtapping.
5.Takeplacethetubesinincubatorat37oCfor2-3hours.
6.Afterincubation,immediatelyadd5μlof6XDyetoeach
tube.
7.Runthesamplesonagarosegelat100-120vcurrent
supplyfor45-60minutes.
8.Observethegelunderauvtransilluminator.
Result:
VisualizetheDNAbandsusingUVtransilluminator.
Lane1:1kbDNAladder
Lane2:ReferenceSample1
Lane3:ReferenceSample2
Lane4:ReferenceSample3
1 2 3 4
8.AIM-PCR-amplificationofSSRfromPlants
GenomicDNA
principle
Simple sequence repeats (SSRs), also known as
microsatellites,aretandem repeatsoftwotofivenucleotide
DNA coresequencessuchas(AT)n,(GT)n,(ATT)n,or
(GACA)nspreadthroughouteukaryoticgenomes.TheDNA
sequencesflankingmicrosatellitesaregenerallyconserved
withinindividualsofthesamespecies,allowingtheselection
ofpolymerasechainreaction(PCR)primersthatwillamplify
theinterveningSSRinallgenotypes.Variationinthenumber
oftandem repeats,n,results in differentPCR product
lengths.Theserepeatsarehighlypolymorphic,evenamong
closelyrelatedcultivars,duetomutationscausingvariation
inthenumberofrepeatingunits.Differentallelescanbe
detectedatalocusbythepolymerasechainreaction(PCR),
using conserved DNA sequences flanking the SSR as
primers.TheSSRassayisbeingincreasinglyappliedtoplant
mappingprojectsduetoitsrelativeadvantages.First,SSRs
arehighlypolymorphicandthushighlyinformativeinplants.
Second,SSRscanbeanalyzedbyarapidandtechnically
simple PCR-based assay.Third,SSRs are co-dominant
markers.Finally,SSRsareboth abundantand uniformly
dispersedinplantgenomes.
Requerements
1.TaqDNApolymerase
2.GenomicDNA(5ng/l)
3.dNTPmix(2mMeachofdATP,dCTP,dGTPanddTTP)
4.MgCl2(25mM)
5.BufferforDNApolymerase
6.10-meroligonuleotideprimers(5M)
7.Steriledistilledwater
8.Electrophoresisgradeagarose
9.0.5XTBEbuffer
10.Ethidiumbromidesolution(10mg/mll)
11.DNAlengthmarker
12.Loadingbuffer
13.PCRmachine
PREPRATIONOFPCRMIX
Buffer10X 500μl
MgCl25mM 300μl
dTTP100mM 10μl
dATP100mM 10μl
dCTP100mM 10μl
dGTP100mM 10μl
ddH20 460μl
Totalvolume 1.3ml
PROCEDURE
1. Set-upa50-μlreactionintolabeledsterile0.25-ml
tubes.
2. PrepareaMIXincludingthePCRMIX,theprimers,
andtheTaqpolymerase.Keepthetubesinice.
3. Addthecomponentsinthefollowingorder:Water,
DNA,MIX(PCRMIX+primers+Taq).Mixwelland
seal
4. tubeswithsuppliedlids.Makesurethelidsareon
tightly.
5. PlacethePCRplatecarryingthereactiontubesin
thesampleblockofthethermocycler.
6. .Carryoutaninitialdenaturationstepat94Cfor
4minfollowedby40cycleswiththefollowingcycle
parameters:
Step194Cfor1min
Step235Cfor1min
Step372Cfor2min
Extendthe72Cstepofthefinalcycleby5min
7. Whentheamplificationhasfinished,add3lofthe
loadingdyetoeachsample.
8. Preparea1.2%agarosegelin0.5XTBEbuffer
containingethidiumbromide(5g/mlofgel).Load
theDNAlengthmarkerandthesamples.Runthegel
in0.5xTBEbufferat55Vfor4h.
RESULT

8. 8
Abhishekkaushik
StaingelinethidiumbromideandVisualizethegelonaUV
transilluminator.