Advantage 2 PCR Enzyme System - Takara1].pdf · 2017-09-12 · Protocol No. PT3281-1 Clontech Laboratories, Inc. Version No. PR621512 3 Advantage® 2 PCR Enzyme System User Manual

Advantage® 2 PCR Enzyme SystemUser Manual

PT3281-1 (PR621512)Published 02 March 2006

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3281-1 2 Version No. PR621512

Advantage® 2 PCR Enzyme System User Manual

Table of Contents

I. Introduction 3

II. List of Components 6

III. Additional Materials Required 7

IV. General Considerations 9

A.PrimerDesign 9

B.TemplateQuality 9

C.AmpliconSize 9

D.BackgroundAmplification 10

E.GoodPCRPractices 10

F.AdditionalConsiderations 11

V. Advantage® 2 PCR Procedure 12

A.ControlPCRReactions 12

B.RecommendedCyclingParametersforallAdvantage2PCRProducts 13

C.RecommendationsforElectrophoresis 14

D.Using10XAdvantage2SAPCRBuffer 14

VI. Troubleshooting Guide 16

VII. References 20

VIII. Related Products 20

Protocol No. PT3281-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR621512 3


I. Introduction

TheAdvantage® 2 Polymerase MixandAdvantage® 2 PCR Kit(whichincludesthePolymeraseMix)produceefficient,accurate,andconvenientamplificationofDNAfromanytemplate.The Advantage 2 Polymerase Mix is comprised of TITANIUM™ Taq DNAPolymerase—anuclease-deficientN-terminaldeletionofTaqDNApolymeraseplus TaqStart™Antibody to provide automatic hot-start PCR (Kellogg et al.,1994)—andaminoramountofaproofreadingpolymerase.TITANIUMTaqprovidesthemostsensitiveandrobustcapabilitiesofanyTaq-de-rivedpolymerase.ItsincreasedsensitivityandrobustnatureareespeciallyusefulforamplifyingawidesizerangeofDNAfragments,cDNAsofraretranscripts,orproductsfromcomplextemplates.ThehigheryieldsandincreasedsensitivitythatTITANIUMTaqprovidestranslateintotwomajoradvantagesoverconventionalpolymerases.First,targetscanbeamplifiedusingfewerPCRcycles,savingtimeandloweringbackgroundinanygivenexperiment.Second,insituationswheretheamplificationtargetispresentatextremelylowlevels(e.g.,amplifyingararecDNAinanRT-PCRexperimentordetectingviralnucleicacid),thehighsensitivityobtainedwithTITANIUMTaqallowssuccessfulamplificationofyourtargetwhereotherpolymerasesfail.TITANIUMTaqallowsyoutoperformPCRwithouttediousbufferoptimization.Inanygivenreaction,TITANIUMTaqtoleratesawiderangeofMg2+concentrationsresultinginhigheryields,especiallyforlongeramplicons.MagnesiumisalreadyincludedatasetconcentrationintheAdvantage 2 PCR Buffer,eliminatingtheneedtoaddMg2+asaseparatecomponentduringreactionsetup.Incontrast,nativeTaqpolymeraseonlyfunctionswelloveranarrow[Mg2+]range,anddif-ferentreactionsmayrequiredifferentconcentrationsofMg2+.ByeliminatingtheneedtoperformexperimentsfordeterminingtheoptimalMg2+concentration,TITANIUMTaqsavesconsiderabletimeandeffort.Incaseswherebackgroundamplification isaproblem,werecommendtheuseof10XAdvantage®2SAPCRBuffer.The10X Advantage® 2 SA PCR Buffer,availableseparately(Cat.No.639147&639148),hasbeenoptimizedforapplicationsusinggenomicDNA,andinparticularwithproductslessthan1-2kb.WehavefoundyieldstobecomparabletothatobtainedwithAdvantage2PCRBufferforrelativelysmallamplicons;however,foramplicons>2kb,werecommendtheuseoftheAdvantage2PCRBufferforbestresults.The10XAdvantage2SAPCRBuffercontainsMg2+ataconcentra-tionoptimizedformostapplications,particularlythoseutilizingagenomicDNAtemplate.Inaddition,theAdvantage2SAPCRBufferisrecommendedwhenbackgroundamplificationispresent,whichmaysometimesresultasaby-productofthesensitivityandrobustnessoftheAdvantage2PolymeraseMix.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3281-1 � Version No. PR621512


I. Introduction continued

The Advantage® Polymerase Systems

ThesimultaneoususeoftwodifferentDNApolymerases(primaryandproofread-ing)inaPCRreactionallowsamplificationofsignificantlylongerfragmentsinaprocessknownaslongandaccuratePCR(orlong-distancePCR[LDPCR];Barnes, 1994; Cheng et al., 1994). However, the usefulness of two-enzymesystemsisnotlimitedtoLDPCR.Infact,theefficiencyofmost PCRreactionscanbesignificantlyimprovedbyusingthetwo-enzymecombination.TheAdvantage2systemoffersthreeprimarybenefitsoverconventional,single-polymerasePCR: — Increased range. Whereas the upper limit of conventional PCR us-

ingaTaqpolymerase is~3kb(andmuch lower inmanyapplications),Advantage2givesconsistentandefficientamplificationsofupto18kbormorewhenusingtwonondegenerateprimersofsufficientlengthtoamplifyanabundant,noncomplextemplate.Itcanalsoamplifyhigh-complexity(i.e.,genomic)DNAtemplatesupto6kb.Theabsoluteupperlimitinanyparticularapplicationwilldependontheparticularprimers,thetemplateused,andotherexperimentalvariables.

—Increased fidelity.Theinclusionofaminoramountofaproofreadingpoly-meraseresultsinanerrorratethatis3-foldlowerthanthatofconventionalPCRusingTaqalone(Barnes,1994;Freyet al.,1995;Nelsonet al.,1995).Inourstudies,Advantage2exhibitsanerrorrateof25errorsper100,000bpafter25PCRcycles.Notethatthepresenceoforganicsolventsorsaltsinthereactioncandecreasefidelity.Highfidelityisaparticularlyimportantfeaturewhentheamplificationproductswillbeusedinsubsequentexperi-ments(e.g.,cloning,sequencing,functionalassays,expressionsystems,etc.).

—Increased efficiency and greater yields.WhilerangeandfidelityarethemostcommonlynotedaspectsoflongandaccuratePCR,theuseofatwo-polymerasesystemalsoincreasestheefficiencyandyield—andthereforethesensitivity—ofall PCRassays,evenfortemplatesthatarewellwithintherangeofconventionalPCR.WhileDNApolymeraseswithproofread-ingactivityofferbetteraccuracythanLDPCRmethodswhenusedalone,theylacktheincreasedefficiencyandsize-rangeflexibilitypossiblewithAdvantage2systemforlongandaccuratePCR.

Automatic hot start with TaqStart™ Antibody

TheAdvantage 2 system contains built-in, hot-start PCR from TaqStartAn-tibody included in the polymerase mix. Antibody-mediated hot start usingTaqStartAntibodyhasbeenshowntosignificantly improvetheefficiencyandspecificityofPCRamplificationsbyreducingbackgroundDNAsynthesis(Kellogget al.,Clontechniques,1994;Kellogget al.,BioTechniques,1994).Specifically,thisantibodyreducesoreliminatesnonspecificamplificationproductsandprimer-dimerartifactscreatedpriortotheonsetofthermalcycling.



I. Introduction continued

TaqStartisaneutralizingmonoclonalantibodythatrecognizesbothnativeTaqandN-terminaldeletionssuchasTITANIUMTaq.TheantibodyinhibitsenzymaticactivityduringPCRreactionsetupatambienttemperatures.Polymeraseactivityisrestoredattheonsetofthermalcyclingbecausetheantibodyisdenaturedathightemperatures.Thelossofinhibitioniscompleteandirreversible,sothepolymeraseregainsitsfullenzymaticactivityforPCR.

Besides increased specificity and sensitivity, the built-in hot start in theAdvantage2PolymeraseMixoffersconvenience.Othermethodsofhotstartrequireextrastepssuchas theadditionandpremeltingofwaxbeadsor theadditionofacriticalcomponentaftertheinitialdenaturation.Theseextrastepsare inconvenient and introduceapotential sourceof cross-contamination. Incontrast,TaqStartprovidesautomatichot-startPCRwithvirtuallynoriskofcross-contamination.Thus,TaqStartAntibodyprovidesalltheadvantagesofhot-startPCRwithnoneofthedisadvantagesofotherhot-startmethods.TheantibodycomesalreadyincludedintheAdvantage2PolymeraseMix;thereisnoneedtoadditasaseparatereagentduringPCRsetup.

Recommended uses for Advantage® 2 Products

The Advantage 2 Mix and Kit are the recommended polymerase systemsfor use in applications involving RACE, RT-PCR, cDNA synthesis and li-brary construction, cDNA subtraction and differential display, high-perfor-mance cloning, and RNA fingerprinting. TheAdvantage 2 System has beenoptimized for use with all Clontech PCR-based application kits, includingSMART™cDNALibraryConstruction,SMART™PCRcDNASynthesis,SMART™RACEcDNAAmplification,ClontechPCR-Select™SubtractionKits,andMara-thon® cDNA Amplification, including GenomeWalker™ Kits, we recommendAdvantage® Genomic Kits. See Related Products (Section VIII) for orderinginformation.



Advantage® 2 PCR Kit (Cat.Nos.639207&639206)Storeallcomponentsat–20°C.Enoughreagentsaresuppliedfor30or100PCRreactionsof50µleach.

639207 639206 30 rxns 100 rxns

• 30 µl 100 µl 50X Advantage® 2 Polymerase Mix IncludesTITANIUMTaqDNAPolymerase,asmallamount

of proofreading polymerase, and TaqStart Antibody(1.1µg/µl)inthefollowingstoragebuffer:

•200 µl 600 µl 10X Advantage® 2 PCR Buffer

•200 µl 600 µl 10X Advantage® 2 SA PCR Buffer• 50 µl 120 µl 50X dNTP Mix(10mMeachofdATP,dCTP,dGTP,anddTTP;

finalrxnconcentration:0.2mMeach)

• 30 µl 100 µl Control DNA Template(100ng/µl) CalfThymusDNA

• 30 µl 100 µl Control Primer Mix(10µMeach) 5’primer5’–GCAACTGCAGGAAGAGCAAGAAATGCA–3’

3’primer5’–TGGCACGGCCATAAGAGGTAGATGTCA–3’

• 2.5 ml 5.0 ml PCR-Grade Water

II. List of Components

Concentrationin50Xmix Component

Finalrxnconcentration

50% Glycerol 1%

15mM Tris-HCl(pH8.0) 0.3mM

75mM KCl 1.5mM

0.05mM EDTA 1.0µM



400mM Tricine-KOH(pH8.7at25°C)

40mM

150mM KOAc 15mM

35mM Mg(OAc)2 3.5mM

37.5µg/ml BSA 3.75µg/ml

0.05% Tween20 0.005%

0.05% Nonidet-P40 0.005%

Protocol No. PT3281-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR621512 �


Advantage® 2 Polymerase Mix (Cat.Nos.639201&639202)Storeallcomponentsat–20°C.Enoughreagentsaresuppliedfor100or500PCRreactionsof50µleach.

639201 639202 100 rxns 500 rxns

• 100 µl 5 x 100 µl 50X Advantage 2 Polymerase Mix Seepage6forcomponentconcentrations.

• 600 µl 5 x 600 µl 10X Advantage 2 PCR Buffer Seepage6forcomponentconcentrations.

• 600 µl 5 x 600 µl 10X Advantage 2 SA PCR Buffer

10X Advantage® 2 SA PCR Buffer (Cat.No.639147&639148)Storeat–20°C.Enoughbufferissuppliedfor240or2,000PCRreactionsof50µleach.

639147 639148 240 rxns 2,000 rxns

• 2 x 600 µl 10 ml 10X Advantage® 2 SA PCR Buffer Bufferalone.

III. Additional materials required

Thefollowingreagentsarenotsupplied.

• Thermal cycler

• Dedicated pipettors (1–2µl,1–10µl,1–20µl,20–200µl,200–1000µl)• PCR pipette tips suitableforusewiththeabovepipettorsandpreferably equippedwithhydrophobicfilters.• DNA size markers (SeeSectionV.D.)• 5X Stop/loading buffer(Sambrook&Russell[2001]providesseveralreci pes)

II. List of Components continued



100mM Tris-HCl(pH8.5) 10mM

500mM KCl 50mM

20mM MgCl2 2mM



• PCR-grade water AvoidusingautoclavedH2Obecauserecycledsteaminsomeautoclaves canintroducecontaminantsthatmayinterferewithPCR.• PCR reaction tubes • [Optional]:Mineral oil(WerecommendSigmaCat.No.M3516.)

IfyouhavepurchasedtheAdvantage 2 Polymerase Mixalone(Cat.Nos.639201&639202),youwillneedthefollowing:• dNTPmix• appropriatecontroltemplateandprimers

IfyouhavepurchasedtheAdvantage 2 SA PCR Bufferalone(Cat.Nos.639147&639148),youwillneedthefollowing:• heat-stableDNAPolymerase• dNTPmix• appropriatecontroltemplateandprimers

III. Additional Materials Required continued

Protocol No. PT3281-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR621512 �


A. Primer Design Primerdesignisthesingle largestvariable inPCRapplicationsandthe singlemostimportantfactorindeterminingthesuccessorfailureofPCR reactions.Always check and recheck your primer design before construct- ing or ordering primers. Length and G-C content: TheAdvantage 2 system can be used in a widevarietyofPCRapplications,andtheconstraintsonprimerdesignwill varyfromoneapplicationtothenext.Ingeneral,however,primersshould haveaTmofapproximately70°Ctoachieveoptimalresultsinatwo-step cyclingprogramwitha68°Cannealing/extensionstep.Therefore,when- everpossible,primersshouldbeat least 22nucleotides(nt)long(25–30- mersarepreferred)andshouldhaveaG-Ccontentof45–60%.Further- more,the3’-terminalendsofeachprimershouldnotbecomplementaryto eachotherandshouldcontainalowG-Ccontent.

B. Template Quality Because PCR amplification proceeds exponentially, many conventional PCRapplicationsworkwellwithtemplatesofaverageorevenlowquality. In many applications (such as screening cDNA inserts with LD Insert Screening Amplimers), long and accurate PCR with Advantage 2 will alsotolerateawiderangeoftemplatequality. However, the longer or more complex the target, the more important templatequalitybecomes.Thisisbecausethenumberofunnicked,full- length targetsdecreasesas the target length increases,sopoor-quality DNA will have very few large, unnicked targets. Furthermore, some depurinationoccurswhenDNAisdenaturedduringthermalcycling,and thiscanleadtotruncatedproducts.Therefore,itisparticularlyimportantto prepare high-quality, high molecular-weight DNA when amplifying largetargets. Templatequalityisalsoimportantwhenthehighestpossiblesensitivityis needed. In cDNA applications such as RACE and RT-PCR protocols, incomplete reverse transcription can lead to an absence of product, truncatedproducts,oramixoftruncatedandfull-lengthproduct,resulting inasmearedbandonagel.Thisproblemcanbeminimizedbyensuringthat yourstartingmaterial isof thehighestquality.For5’and3’RACEand generalPCRfromcDNA,youcanensurethequalityofyourcDNAbyusing Marathon-ReadycDNAfromClontech.

C. Amplicon Size Yieldsareunaffectedwhenamplifyingproductslessthan2kb,whetherthe 10X Advantage 2 SA PCR Buffer or the Advantage 2 PCR Buffer is used. We recommend initial use of the 10XAdvantage 2 PCR Buffer; however,ifaproblemarises,inparticularwithampliconslessthan2kbin length, then we recommend switching to the 10X Advantage 2 SA PCRBuffer.Forproductsgreaterthan2kb,werecommendtheoriginal

IV. General Considerations



IV. General Considerations continued

Advantage2PCRBufferbeused(ratherthantheSAPCRBuffer). D. Background Amplification Due to the sensitivity and robustness of theAdvantage 2 Polymerase Mix, non-specific background amplification may sometimes result. The 10XAdvantage2ShortAmplicon(SA)PCRBufferhasbeenspecifically engineeredtoaddressthisissue.Werecommendtheuseofthisbufferin thoseapplicationswheresomebackgroundamplificationmayresult. E. Good PCR Practices 1.Preparereactionswithdedicatedpipettorsinadedicatedworkspace DuetothetremendousamplificationpowerofPCR,minuteamounts

ofcontaminatingDNAcanproducenonspecificamplification;insomeinstances,contaminantscancauseDNAbandsevenintheabsenceofaddedtemplateDNA.Werecommendthatyouusesmallaliquotsofstartingmaterialtoavoidcontaminatingyourstocks.WhenperformingPCR,youshouldwearglovesandsetupyourreactionsinadedicatedlabareaornoncirculatingcontainmenthoodusingdedicatedpipettors,PCRpipettetipswithhydrophobicfilters,anddedicatedsolutions.Wealsorecommendsettingupanegativecontrolreactionthatdoesnotcontainanytemplate.Finally,performpost-PCRanalysisinaseparateareausingaseparatesetofpipettors.

2.Pipetting Because of the small volumes used in PCR experiments and the

potential for tube-to-tube variation, careful pipetting technique isextremelyimportant.Alwaysbesurethatnoextrasolutionisontheoutside of the pipette tip before transfer. When adding solution toa tube, immerse the tip into the reaction mixture, deliver the con-tents from the pipette tip into the mixture, and pipet up and downseveraltimes.

3.UseaMasterMix Assembling a Master Mix, which contains the appropriate volumes

ofall reagents required formultiplePCRreactions,saves timeandgreatlyreducestube-to-tubevariation.Ifmultipletemplatesarebeingtestedwiththesameprimers,includetheprimersintheMasterMix.Ifonetemplateisbeingtestedwithmultipleprimersets,includethetemplateintheMasterMix.Ifyouaresettingupseveralsetsofparallelsamples,assemblemultipleMasterMixes(e.g.,eachwithadifferentsetofprimers).The Master Mix should be thoroughly mixed before use (i.e., vortexed without bubbling).

4.Alwaysincludepositiveandnegativecontrols(i.e.,H2OinsteadofDNAtemplate).



F. Additional Considerations 1.TouchdownPCR “Touchdown” PCR can significantly improve the specificity of many

PCRreactionsinawidevarietyofapplications(Donet al.,1991;Roux,1995).Briefly,touchdownPCRinvolvesusinganannealing/extensiontemperaturethatisseveraldegrees(typically3–10°C)higherthantheTmoftheprimersduringtheinitialPCRcycles(typically5–10cycles).Theannealing/extensiontemperatureisthenreducedtotheprimerTmfortheremainingPCRcycles.

2.TaqStartAntibodyprovidesautomatichotstartPCR Theuseofamanualhotstartorwaxbead-basedhotstartisnotrequired

whenusingtheAdvantage2system.AsdiscussedintheIntroduction,hotstartisautomaticbecausetheenzymemixalreadycontainsTaqStartAntibody.

3.Half-life Thehalf-lifeofTITANIUMTaqdependsonthespecificreactioncondi-

tionsused,butgenerallyrangesfrom20–40minat95°C. 4.Useofadditives TaqStartAntibody bindsTITANIUM Taq DNA Polymerase with high

affinityundertheconditionsdescribedinthisprotocol.Theadditionof2–5%DMSOwillnotinterferewithTaqStartantibodyfunctionandmayimproveresultsinsomeinstances.However,theadditionofformamideorothercosolventsmaydisruptTaqStartantibodyfunction.Further-more,excessiveglycerol,solutes(e.g.,salts),pHextremes,orotherdeviationsfromtherecommendedreactionconditionsmayreducetheeffectivenessoftheantibodyand/orDNApolymerases.

5.Advantage2PolymeraseMixisnotintendedforcertainapplications Because of the improved fidelity from long and accurate PCR,

Advantage2isnotrecommendedformutagenesisprotocolsinvolvingso-called“sloppy”PCR.

6.T/ACloning TITANIUM Taq PCR products are compatible with T/A cloning

methods. However, for optimal ligation efficiencies, we recommendthatyouusePCRproductsimmediately(<1day)afteramplification.The single3’A-overhangson thePCRproductswill degradeovertime,reducingtheefficiency.Forbestresults,pleaseobservetheT/AcloningtipsinSectionV.C.

IV. General Considerations continued



PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING.

A. Control PCR Reactions ThefollowingcontrolPCRreactionsshouldbeperformedinparallelwith

yourexperimentstoensurethattheAdvantage2PolymeraseMixiswork-ingproperly.ApositivecontroltemplateandprimersareprovidedintheAdvantage2PCRKit.WhenusingtheAdvantage2PolymeraseMixwithourotherPCR-basedapplicationskits,usethepositivecontrolsprovidedwiththosekits.

1.Place all components on ice and allow to thaw completely. Mixeachcomponentthoroughlybeforeuse.

2.CombinethefollowingreagentsinaPCRtube.

3.Mixwellandspintubebrieflytocollectalltheliquidinbottomoftube. 4.[Optional]: Ifyour thermalcyclerdoesnothavea “hot lid”,add1–2

dropsofmineraloiltopreventevaporationduringcycling.Agood“seal”ofmineraloilshouldhaveawell-definedmeniscusbetweenthetwophases.CapthePCRtubesfirmly.

5.Commencethermalcyclingusingthefollowingparameters: •95°Cfor1min •30cycles* 95°Cfor15sec 68°Cfor3min

*30cycleswitha3-minannealing/extensiontimeissufficientforamplificationofthepositivecontroltemplateprovidedinthekit.Othertemplatesmayrequiremoreorlesscyclesanddifferentannealing/extensiontimes(SeeSectionV.B.).

6.Transfera5µlsampleofyourPCRreactiontoafreshtubeandadd1µlof5Xstop/loadingbuffer.Analyzeyoursample(s),alongwithsuitableDNAsizemarkers,byelectrophoresisona1.2%agarose/EtBrgel.

Expected results: Ifyouareusingthepositivecontrolreagentsprovidedinthekit,thereactionshouldproduceasinglemajorfragmentof3.5kb,

V. Advantage® 2 PCR Procedure

PositiveControl

NegativeControl

40µl 41µl PCR-GradeWater

5µl 5µl 10XAdvantage2PCRBuffer

1µl --- ControlDNATemplate(100ng/µl)

2µl 2µl ControlPrimerMix(10µMea.)

1µl 1µl 50XdNTPMix(10mMea.)

1µl 1µl 50XAdvantage2PolymeraseMix

50 µl 50 µl Total volume



derivedfromthegeneforthebovinepancreatictrypsininhibitor.Nobandsshouldbegeneratedinthenegative(i.e.,noDNAtemplate)control.

B. Recommended Cycling Parameters for all Advantage® 2 PCR Products

UsethefollowingguidelineswhensettingupyourinitialexperimentswiththeAdvantage2system.Thesearegeneralguidelines—theoptimalpa-rametersmayvarywithdifferentthermalcyclersandwilldependonyourparticularprimers,template,andotherexperimentalvariables.

Notes: • WhenusingtheAdvantage2PolymeraseMixwithourotherPCR-based

application kits (suchasSMARTcDNALibraryConstruction,SMARTPCRcDNASynthesis,ClontechPCR-SelectSubtractionKits,MarathoncDNA Amplification, or Marathon-Ready cDNAs, use the parametersrecommendedintheprotocolforthatkit.

• Ifyou intend tocaptureyourPCRproductbyT/Acloning,werecom-mend thatyouaddanadditional10-minextensionat70°C,and thenimmediatelycloneorfreezethePCRproduct.Donotstorethereactionat4°C.Thesestepswillhelpensuretheincorporationandpreservationof3’A-overhangs.

Target Cycle Size Parameters

<1kb: •95°Cfor1min •25–35cyclesa

95°Cfor30secb

68°Cfor1minc

•68°Cfor1mind

1–5kb: •95°Cfor1min •25–35cyclesa

95°Cfor30secb

68°Cfor3minc

•68°Cfor3mind


95°Cfor30secb

68°Cfor6minc

•68°Cfor6mind

V. Advantage® 2 PCR Procedure continued

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3281-1 1� Version No. PR621512



95°Cfor30secb

68°Cfor12minc

•68°Cfor12mind

a 25 cycles for multiple-copy genes or medium-to-high abundance cDNAs; 30–35cyclesforsingle-orlow-copy-numbergenesorrarecDNAs.Formostapplications,weprefertwo-stepcycles(denaturationatT1followedbyannealingandextensionatT2)insteadofthree-stepcycles(denaturationatT1followedbyannealingatT2followedbyextensionatT3).Three-stepcycleswillbenecessarywhentheTmoftheprimersislessthan60–65°Candincertainspecialprotocols.

b Usetheminimalpossibledenaturationtime.Insomecases,betterresultsmaybeob-tainedbymodifyingthedenaturationstep(94°Cfor15sec).ExposureofDNAtohightemperaturescausessomedepurinationofsingle-strandedDNAduringdenaturation,whicheventuallyleadstostrandscission.Hightemperaturealsoleadstograduallossofenzymeactivity.Minimizingdenaturationtimeisparticularlyimportantinexperimentswithverylargetemplateswheretotalcyclingtimecanexceed12hr.

c Usethemaximumpossibleannealing/extensiontemperature.See“Notea”.Someresearchersprefertouseanannealing/extensiontimeequaltotheexpectedtargetsize(inkb)plustwominutes.Werecommendusing1minperkbofexpectedtarget.

d Optional:Thisfinalextensionmayreducebackgroundinsomecases.

C. Recommendations for Electrophoresis Werecommendthatyoutransfera5µlsampleofyourPCRreactionto

afreshtubeandadd1µlof5Xstop/loadingbuffer.Placetheremaining45µlofthereactionmixtureonice;itcanbesubjectedtofurthercyclingifyoudonotseeaproduct.Analyzeyoursample(s),alongwithsuitableDNAsizemarkers,byelectrophoresisonasuitableagarosegelcontaining0.1µg/mlEtBr.ThepercentageagaroseandtheDNAsizemarkersyouchoosewilldependontheexpectedrangeofinsertsizes.Youmaywishtorefertothefollowinggeneralguidelinesbeforeassemblingyourgel.

Recommendationsforagarosegels:

D. Using 10X Advantage 2 SA PCR Buffer Incaseswherenon-specificbackgroundamplificationisaproblem,we

stronglyrecommendthe10XAdvantage2SAPCRBufferbeusedasthebufferofchoice.


Expectedinsertsizerange

Recommended%agarose

RecommendedDNAsizemarkers

0.3–1.5kb 1.5 φX174/Hae III

0.5–10kb 1.2 1kbDNAladder

>5kb 0.8 λ/HindIII



Yieldsareunaffectedwhenamplifyingproducts lessthan2kb,whetherthe10XAdvantage2SAPCRBufferor theAdvantage2PCRBuffer isused.Werecommendinitialuseofthe10XAdvantage2PCRBuffer;andifbackgroundamplificationarises, inparticularwithampliconslessthan2kbinlength,thenwerecommendswitchingtothe10XAdvantage2SAPCRBuffer.




The followinggeneral guidelinesapply tomostPCR reactions.However,noattempthasbeenmadetoaddresstroubleshootingforallofthemanyapplica-tionsforwhichtheAdvantage2PolymeraseMixcanbeused.WhenusingtheenzymewithoneofClontechcompanionproducts,additionalapplication-specifictroubleshootinginformationcanbefoundintherelevantUserManual.

A. No product observed PCRcomponent Useachecklistwhenassemblingreactions.Always missingordegraded performapositivecontroltoensurethateachcom-

ponentisfunctional.Ifthepositivecontroldoesnotwork,repeatthepositivecontrolonly.Ifthepositivecontrol still does not work, repeat again replacingindividualcomponentstoidentifythefaultyreagent.

Toofewcycles Increasethenumberofcycles(3–5additionalcyclesatatime).

Annealingtemp. Decreasetheannealingtemperatureinincrements toohigh of2–4°C.

Suboptimalprimer Redesignyourprimer(s)afterconfirmingtheaccu- design racy of the sequence information. If the origi-

nal primer(s) was less than 22 nt long, try us-ing a longer primer. If the original primer(s) hada G-C content of less than 45%, try to designaprimerwithaG-Ccontentof45–60%.

Notenough RepeatPCRusingahigher concentrationofDNA template (aftertryingmorecycles).

Poortemplate Check template integrity by electrophoresis on a quality standardTBE-agarosegel.Ifnecessary,repurifyyour

templateusingmethodsthatminimizeshearingandnicking.

Denaturationtemp. Optimizedenaturationtemperaturebydecreasingor toohighorlow increasingitin1°Cincrements.(Adenaturationtem-

peraturethatistoohighcanleadtodegradationofthetemplate,especiallyforlongtargetsequences.)

Denaturationtime Optimizedenaturationtimebydecreasingorincreas- toolongortooshort ingitin10secincrements.(Adenaturationtimethat

istoolongcanleadtodegradationofthetemplate,especiallyforlongtargetsequences.)

Extensiontimetoo Especially for longer templates, increase the short extensiontimein1minincrements.

VI. Troubleshooting Guide

Protocol No. PT3281-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR621512 1�


Toolittleenzyme TheAdvantage2PolymeraseMixis50Xformostap-plications;howevera1Xfinalreactionconcentrationoftheenzymemixmaybetoolowforsomeapplications.Therefore, try tooptimize thecycleparametersasdescribedabovebeforeincreasingtheenzymecon-centration.Inrarecases,theyieldscanbeimprovedbyincreasingtheconcentrationoftheenzymemix.However,increasingthefinalconcentrationto>2Xinthereactionmixislikelytoleadtohigherbackgroundlevels.

[Mg2+]istoolow TheAdvantage2PolymeraseMixperformswellatabroadrangeofMg2+concentration.Therefore,aslongasyouusethebufferincludedwiththemixandafinalconcentrationof0.2mMofeachdNTP,it isunlikelythatalackofproductisduetoproblemswiththeMg2+concentration.However,highconcentrationsofEDTAorothermetalchelatorsinthetemplatestocksolution can reduce the effective concentration ofMg2+tobelowaminimumlevel.

[dNTPs]istoolow When used as recommended, the 50X dNTP mixprovidedwith thekitgivesafinal concentrationof0.2mMofeachdNTP.Inourexperience,thiscon-centrationofdNTPsissuitableforawiderangeofapplications.

IfyouarepreparingyourowndNTPs,besurethatyourfinalconcentrationofeachdNTPinthereactionis0.2mM.

Somemanufacturersrecommendusingconcentra-tionshigherthan0.2mMofeachdNTPwhenam-plifyinglargetemplates.However,wehavehadnotroubleamplifyinglargetemplatesusing0.2mMforeachdNTP.Sincewehavegoneupto35kbwiththeAdvantageGenomicPCRKit,itisunlikelythatdNTPsarelimiting.NotethatifyouincreasetheconcentrationofdNTPs,youwillalsoneedtoincreasethe[Mg2+]proportionately.

Difficulttarget Sometargetsareinherentlydifficulttoamplify.Inmostcases,thisisduetounusuallyhighG-Ccontentand/orsecondarystructure.UseAdvantageGCPolymeraseMixorKitinsteadoftheAdvantage2system.

VI. Troubleshooting Guide continued



B. Multiple products Toomanycycles Reducingthecyclenumbermayeliminatenonspecific

bands.

Annealingtemp. Increase the annealing/extension temperature in toolow incrementsof2–3°C. Suboptimalprimer Redesignyourprimer(s)afterconfirmingtheaccu- design racy of the sequence information. If the original

primer(s)waslessthan22ntlong,tryusingalongerprimer. If theoriginalprimer(s)hadaG-Ccontentoflessthan45%,trytodesignaprimerwithaG-Ccontentof45–60%.

TouchdownPCR “Touchdown”PCRsignificantlyimprovesthespeci- needed ficityofmanyPCRreactionsinvariousapplications

(Donet al.,1991;Roux,1995).TouchdownPCRin-volvesusinganannealing/extensiontemperaturethatisseveraldegreeshigherthantheTmoftheprimersduringtheinitialPCRcycles.Theannealing/extensiontemperatureisthenreducedtotheprimerTmfortheremainingPCRcycles.Thechangecanbeperformedeitherinasinglesteporinincrementsoverseveralcycles.

Contamination SeeSectionEbelow.

C. Low yield Poortemplate Check template integrity by electrophoresis on a quality standardTBE-agarosegel.Ifnecessary,repurifyyour

templateusingmethodsthatminimizeshearingandnicking.

D. Products are smeared on gel Toomanycycles Reducethecyclenumberby3–5toseeifnon-specific

bandsgoaway.

Denaturationtemp. Tryincreasingthedenaturationtemperatureinincre- toolow mentsof1°C.

Extensiontime Decreasetheextensiontimein1–2-minincrements. toolong

Poortemplate Checktemplateintegritybyelectrophoresisonade- quality denaturing agarose gel. Repurify your template if

necessary.

TouchdownPCR See “Touchdown PCR needed” under previous needed section.


Protocol No. PT3281-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR621512 1�


Non-specific Use the 10X Advantage SA PCR Buffer priming whichhasbeenspecificallyengineeredtoaddress

this(ratherthanthe10XAdvantage2PCRBuffer).

Toomuchenzyme TheAdvantage2PolymeraseMix is50X formostapplications;however,a1Xfinalconcentrationoftheenzymemixmaybetoohighforsomeapplications.Ifsmearingisobserved,firsttryoptimizingthecycleparametersasdescribedabove, then try reducingtheenzymeconcentrationto0.5–0.2X.

Toomuchtemplate TryalowerconcentrationofDNAtemplateinthePCRreaction.

Contamination SeeSectionEbelow.

E. Dealing with contamination Contaminationmostoftenresultsinextrabandsorsmearing.Itisimportant

toincludeanegativecontrol(acontrolthatreplacestheDNAtemplatewithPCR-gradeH2Obutstillincludestheprimers)ineveryPCRexperimenttodetermineifthePCRreagents,pipettorsorPCRreactiontubesarecon-taminatedwithpreviouslyamplifiedtargets.

Ifpossible,setupthePCRreactionandperformthepost-PCRanalysisinseparatelaboratoryareaswithseparatesetsofpipettors.

Laboratorybenchesandpipettorshaftscanbedecontaminatedbydepuri-nation.Wipesurfaceswith1NHClfollowedby1NNaOH.Thenneutralizewithaneutralbuffer(e.g.,TrisorPBS)andrinsewithddH2O.

Weadviseusingcommerciallyavailableaerosol-freepipettetips. AnenzymaticmethodhasbeenpublishedfordestroyingPCRproductcar-

ryover(Longoet al.,1990).ItinvolvesincorporationofdUTPintothePCRproductsandsubsequenthydrolysiswithuracil-N-glycosylase(UNG).

WhenperformingPCRdirectlyonphageplaquesorbacterialcolonies,failuretoisolatesingleplaquesorcolonieswillalsoproducemultiplebands.




ForacompletelistingofallClontechproducts,pleasevisitwww.clontech.com

PCR Products Cat. No.

• Sprint™TITANIUM™Taq384Plate 639552

• Sprint™Advantage®96Plate 639550

• Sprint™Advantage®SingleShots 639556 639553 639554

• 10XAdvantage2PCRBuffer 639137 639138

• TITANIUM™TaqDNAPolymerase 639208 639209

• TITANIUM™TaqPCRKit 639211 639210

• Advantage®GenomicPolymeraseMix 639110

VII. References

Barnes,W.M.(1994)PCRamplificationofupto35-kbDNAwithhighfidelityandhighyieldfromλbacteriophagetemplates.Proc. Natl. Acad. Sci. USA 91:2216–2220.

Cheng,S.,Fockler,C.,Barnes,W.M.&Higuchi,R.(1994)EffectiveamplificationoflongtargetsfromclonedinsertsandhumangenomicDNA.Proc. Natl. Acad. Sci. USA 91:5695–5699.

Don,R.H.,Cox,P.T.,Wainwright,B.J.,Baker,K.&Mattick,J.S.(1991)‘Touchdown’PCRtocir-cumventspuriousprimingduringgeneamplification.Nucleic Acids Res. 19:4008.

Frey,B.&Suppmann,B.(1995)DemonstrationoftheExpand™PCRsystem’sgreaterfidelityandhigheryieldswithalacI-basedPCRfidelityassay.Biochemica2:8–9.

Kellogg, D. E., Rybalkin, I., Chen, S., Mukhamedova, N., Vlasik,T., Chenchik,A. & Siebert, P.(1994)TaqStart:ANeutralizingMonoclonalAntibodythatfacilitates“HotStart”PCR.Clontechniques IX(2):1–5.

Kellogg,D.E.,Rybalkin,I.,Chen,S.,Mukhamedova,N.,Vlasik,T.,Siebert,P.&Chenchik,A.(1994)TaqStartAntibody:HotstartPCRfacilitatedbyaneutralizingmonoclonalantibodydirectedagainstTaq DNApolymerase.BioTechniques 16:1134–1137.

Longo,M.C.,Berninger,M.S.&Hartley,J.L.(1990)UseofuracilDNAglycosylasetocontrolcarry-overcontaminationinpolymerasechainreactions.Gene93:3749.

Nelson,K.,Brannan, J.&Kretz,K., (1995)The fidelity ofTaqPlus™DNAPolymerase inPCR.Strategies Mol. Biol. 8:24–25.

Roux,K.H.(1995)OptimizationandtroubleshootinginPCR.PCR Methods Appl.4:5185–5194.

Sambrook,J.&D.W.(2001)Molecular Cloning: A Laboratory Manual, Third Edition (ColdSpringHarborLaboratory,ColdSpringHarbor,NY).

VIII. Related Products



VIII. Related Products continued

PCR Products continued Cat. No.

• Advantage®GenomicPCRKit 639104 639103

• Advantage®GC2PolymeraseMix 639114

• Advantage®GC2PCRKit 639120 639119

• Advantage®GCGenomicPolymeraseMix 639113

• Advantage®GCGenomicPCRKit 639118 639117

• Advantage®HF2PCRKit 639124 639123

• Advantage®UltraPurePCR DeoxynucleotideMix 639125

• Advantage®UltraPure dNTPCombinationKit 639132

• TaqStart®Antibody 639250 639251 PCR Amplification Kits & Products

• SMART™cDNALibraryConstructionKit 634901

• SuperSMART™PCRcDNASynthesisKit 635000

• SMART™RACEcDNAAmplificationKit 634914

• Marathon®cDNAAmplificationKit 634913

• Marathon-ReadycDNAs many

• ClontechPCR-Select™ cDNASubtractionKit 637401

• ClontechPCR-Select™BacterialGenome SubtractionKit 637404• LD-InsertScreeningAmplimerSets many• RT-PCRAmplimerSets many

• Advantage®RT-for-PCRKit 639505 639506

• MTC™MultipleTissuecDNAPanels many

• QUICK-Clone™cDNAs many• TITANIUM™One-StepRT-PCRKit 639503 639504



Notes



Notes

Notice to PurchaserThisproduct is intended tobeused for researchpurposesonly. It isnot tobeused fordrugordiagnostic purposes nor is it intended for human use. Clontech products may not be resold,modified for resale, or used to manufacture commercial products without written approval ofClontechLaboratories,Inc..

Advantage®2productsarecoveredbyU.S.PatentNo.5,436,149.

Clontech™PCR-Select™cDNASubtractionproductsarecoveredbyU.S.PatentNos.5,565,340and5,759,822,aswellaspendingforeignpatentapplications.For-profitandnot-for-profitpurchas-ersofPCR-Selectproductsareentitled touse thereagents for research, including identificationofmolecularmarkersordifferentiallyexpressedgenes;however,thefollowingusesexpresslyareprohibited: (1)performingservices for thirdparties; (2) identifyingnucleicacid sequences tobeincludedonnucleicacidarrays,blots,orinlibrariesorothercDNAcollectionswhicharethensoldtothirdparties;or(3)constructingdatabaseswhicharethensoldtothirdparties.Further,reproduction,amplification,modification,reformulation,orresaleofthereagentsprovidedinPCR-Selectproductsisnotpermitted.ForinformationonlicensingPCR-Selectforthesepurposes,pleasecontact:ClontechLaboratories,Inc.,BusinessDevelopment,1290TerraBellaAvenue,MountainView,CA,94043orcalltheLicensingHotlineat650.919.7320ore-maillicensing@clontech.com.

TaqStart®AntibodyislicensedunderU.S.PatentNo.5,338,671.AlicenseunderU.S.patentNos.4,683,202,4,683,195and4,965,188ortheirforeigncounterpartsownedbyRocheMolecularSystems, Inc.andF.Hoffmann-LaRocheLtd. (“Roche”), foruse inresearchanddevelopment,hasanup-frontfeecomponentandarunning-royaltycomponent.BypurchasingcertainAppliedBiosystemsPCRreagents,purchasersobtainlimited,nontransferablerightsundertherunningroyaltycomponenttousethepurchasedamountofreagentstopracticethePolymeraseChainReaction(PCR)andrelatedprocessesdescribedinsaidpatentsonlyforresearchanddevelopmentactivitiesofthepurchaser.Suchuseisfullylicensedunderthesepatentsonlywhenthereagentsareusedinconjunctionwithathermalcyclerwhoseuseiscoveredbytheup-frontfeecomponent.Rightsundertheup-frontfeecomponentofaPCRlicensemaybepurchasedfromAppliedBiosystemsorobtainedbypurchasinganAuthorizedThermalCycler.NorighttoperformoroffercommercialservicesofanykindusingPCR,including,forexample,reportingtheresultsofpurchaser’sactivitiesforafeeorothercommercialconsideration,isgrantedunlessthepurchaserobtainsafurtherlicensefromeitherAppliedBiosystemsorRoche.Additionalinformationonpurchas-inglicensestopracticethePCRprocessmaybeobtainedbycontactingtheDirectorofLicensingatAppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404orRocheMolecularSystems,1145AtlanticAvenue,Alameda,California,94501.

GeneAmp®isaregisteredtrademarkofRocheMolecularSystems,Inc., licensedtotheAppleraCorporation.Clontech,ClontechlogoandallothertrademarksarethepropertyofClontechLaboratories,Inc.ClontechisaTakaraBioCompany.©2006

Documents

Advantage 2 PCR Enzyme System - Takara1].pdf · 2017-09-12 · Protocol No. PT3281-1 Clontech Laboratories, Inc. Version No. PR621512 3 Advantage® 2 PCR Enzyme System User Manual