10/22/2016

PCRProtocolforTaqDNAPolymerasewithStandardTaqBuffer(M0273)|NEB

Home Protocols PCRProtocolforTaqDNAPolymerasewithStandardTaqBuffer(M0273)

PCRProtocolforTaqDNAPolymerasewithStandardTaqBuffer
(M0273)
Protocols.ioalsoprovidesaninteractiveversionofthisprotocolwhereyoucandiscoverandshareoptimizationswiththe
researchcommunity.

Overview
PCR
ThePolymeraseChainReaction(PCR)isapowerfulandsensitivetechniqueforDNAamplification(1).TaqDNAPolymeraseisanenzyme
widelyusedinPCR(2).ThefollowingguidelinesareprovidedtoensuresuccessfulPCRusingNEB'sTaqDNAPolymerase.These
guidelinescoverroutinePCR.AmplificationoftemplateswithhighGCcontent,highsecondarystructure,lowtemplateconcentrations,or
ampliconsgreaterthan5kbmayrequirefurtheroptimization.

Protocol
Reactionsetup:
Werecommendassemblingallreactioncomponentsoniceandquicklytransferringthereactionstoathermocyclerpreheatedtothe
denaturationtemperature(95C).
Component

25lreaction

50lreaction

FinalConcentration

10XStandardTaqReactionBuffer

2.5l

5l

1X

10mMdNTPs

0.5l

1l

200M

10MForwardPrimer

0.5l

1l

0.2M(0.051M)

10MReversePrimer

0.5l

1l

0.2M(0.051M)

TemplateDNA

variable

variable

<1,000ng

TaqDNAPolymerase

0.125l

0.25l

1.25units/50lPCR

Nucleasefreewater

to25l

to50l

Notes:Gentlymixthereaction.Collectallliquidtothebottomofthetubebyaquickspinifnecessary.Overlaythesamplewithmineraloilif
usingaPCRmachinewithoutaheatedlid.
TransferPCRtubesfromicetoaPCRmachinewiththeblockpreheatedto95Candbeginthermocycling.
ThermocyclingconditionsforaroutinePCR:
STEP
InitialDenaturation
30Cycles

FinalExtension
Hold

TEMP

TIME

95C

30seconds

95C
4568C
68C

1530seconds
1560seconds
1minute/kb

68C

5minutes

410C

GeneralGuidelines:
1.Template:
Useofhighquality,purifiedDNAtemplatesgreatlyenhancesthesuccessofPCR.RecommendedamountsofDNAtemplatefora50l
reactionareasfollows:
DNA

Amount

genomic

1ng1g

plasmidorviral

1pg1ng

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PCRProtocolforTaqDNAPolymerasewithStandardTaqBuffer(M0273)|NEB

2.Primers:
Oligonucleotideprimersaregenerally2040nucleotidesinlengthandideallyhaveaGCcontentof4060%.Computerprogramssuch
asPrimer3(http://frodo.wi.mit.edu/primer3)canbeusedtodesignoranalyzeprimers.Thefinalconcentrationofeachprimerina
reactionmaybe0.051M,typically0.10.5M.
3.Mg++andadditives:
Mg++concentrationof1.52.0mMisoptimalformostPCRproductsgeneratedwithTaqDNAPolymerase.ThefinalMg++concentration
in1XStandardTaqReactionBufferis1.5mM.Thissupportssatisfactoryamplificationofmostamplicons.However,Mg++canbe
furtheroptimizedin0.5or1.0mMincrementsusingMgCl2.
Amplificationofsomedifficulttargets,likeGCrichsequences,maybeimprovedwithadditives,suchasDMSO(3)orformamide(4).
4.Deoxynucleotides:
ThefinalconcentrationofdNTPsistypically200Mofeachdeoxynucleotide.
5.TaqDNAPolymeraseConcentration:
WegenerallyrecommendusingTaqDNAPolymeraseataconcentrationof25units/ml(1.25units/50lreaction).However,theoptimal
concentrationofTaqDNAPolymerasemayrangefrom550units/ml(0.252.5units/50lreaction)inspecializedapplications.
6.Denaturation:
Aninitialdenaturationof30secondsat95CissufficientformostampliconsfrompureDNAtemplates.Fordifficulttemplatessuchas
GCrichsequences,alongerinitialdenaturationof24minutesat95CisrecommendedpriortoPCRcyclingtofullydenaturethe
template.WithcolonyPCR,aninitial5minutedenaturationat95Cisrecommended.
Duringthermocyclinga1530seconddenaturationat95Cisrecommended.
7.Annealing:
Theannealingstepistypically1560seconds.AnnealingtemperatureisbasedontheTmoftheprimerpairandistypically4568C.
AnnealingtemperaturescanbeoptimizedbydoingatemperaturegradientPCRstarting5CbelowthecalculatedTm.TheNEBTm
Calculatorisrecommendedtocalculateanappropriateannealingtemperature.
Whenprimerswithannealingtemperaturesabove65Careused,a2stepPCRprotocolispossible(see#10).
8.Extension:
Therecommendedextensiontemperatureis68C.Extensiontimesaregenerally1minuteperkb.Afinalextensionof5minutesat
68Cisrecommended.
9.Cyclenumber:
Generally,2535cyclesyieldssufficientproduct.Upto45cyclesmayberequiredtodetectlowcopynumbertargets.
10.2stepPCR:
Whenprimerswithannealingtemperaturesabove65Careused,a2stepthermocyclingprotocolispossible.
Thermocyclingconditionsforaroutine2stepPCR:
STEP
InitialDenaturation

TEMP
95C

TIME
30seconds

30Cycles

95C
6568C

1530seconds
1minute/kb

FinalExtension

6568C

5minutes

Hold

410C

11.PCRproduct:
ThePCRproductsgeneratedusingTaqDNAPolymerasecontaindAoverhangsatthe3endthereforethePCRproductscanbe
ligatedtodT/dUoverhangvectors.
References:
1.SaikiR.K.etal.(1985).Science.230,13501354.
2.Powell,L.M.etal.(1987).Cell.50,831840.
3.Sun,Y.,Hegamyer,G.andColburn,N.(1993).Biotechniques.15,372374.
4.Sarkar,G.,Kapelner,S.andSommer,S.S.(1990).NucleicAcidsRes..18,7465.

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