1sjnf453 (9- %/1pmznfsbtf · (1) primer design select primer sequences using primer design software...

Cat. # R050A

Product Manual

PrimeSTAR® GXLDNA Polymerase

For Research Use

v201906Da

Table of Contents

I. Description...................................................................................................................3

II. Components................................................................................................................3

III. Storage..........................................................................................................................3

IV. Protocols........................................................................................................................4

V. OptimizationofParameters..................................................................................6

VI. Electrophoresis,Cloning,andSequencingofAmplifiedProducts........7

VII. Troubleshooting.........................................................................................................7

VIII. Features.........................................................................................................................8

IX. RelatedProducts..................................................................................................... 11

2

PrimeSTAR® GXL DNA PolymeraseCat. #R050A

v201906Da

URL:http://www.takara-bio.com

I. DescriptionPrimeSTARGXLDNAPolymeraseisarevolutionaryPCRenzymethataugmentsthehigh-fidelityPrimeSTARHSDNAPolymerasewithanovelelongationfactortodramaticallyincreasePCRperformance.ThesuperiorperformanceofPrimeSTARGXLDNAPolymeraseisunsurpassedbyothercommerciallyavailablehigh-fidelityPCRenzymes.PrimeSTARGXLDNAPolymeraseallowsamplificationofproducts≧30kbinlength,whilemaintainingexceptionallyhighfidelity.SuitableforGC-richtemplatesthatareotherwisedifficulttoamplify,thisenzymeenablessuccessfulamplificationfromchallengingtemplateswithouttheneedforextensiveoptimizationofbuffersorreactionconditions.Inaddition,PrimeSTARGXLDNAPolymeraseiscompatiblewithawiderangeoftemplatequantities,andiscapableofrobustamplificationeveninthepresenceoflargeexcessesofnon-targetDNA.Excessnon-targetnucleicacidtypicallyinhibitstheperformanceofconventionalhigh-fidelityPCRenzymes.WithPrimeSTARGXLDNAPolymerase,detectionofcDNAcorrespondingtoraretranscriptsisreadilyachieved.Furthermore,anantibody-mediatedhot-startformulationpreventsfalseinitiationeventsduringreactionassemblyduetomisprimingorprimerdigestion.ThePCRextensiontimerecommendedintheStandardProtocolis1min/kb.However,bydoublingthequantityofenzymeused,itisalsopossibletoperformrapidPCRonawidevarietyoftargetsusinganextensiontimeof10sec/kb.

II. Components (200 reactions)＊1

PrimeSTARGXLDNAPolymerase(1.25U/μl)＊2 200μl5XPrimeSTARGXLBuffer(Mg2+plus)＊3 1mlx2dNTPMixture（2.5mMeach） 800μl

＊1 Assuminga50μlreactionvolume

＊2【StorageBuffer】 50mM Tris-HCl(pH8.2at4℃ ) 100mM NaCl 0.1mM EDTA 1mM DTT 0.1% Tween20 0.1% NP-40 50% Glycerol

【Unitdefinition】Oneunitistheamountofenzymethatwillincorporate10nmolofdNTPintoacid-insolubleproductsin30minutesat74℃withactivatedsalmonspermDNAasthetemplate.

＊3 Mg2+concentration:5mM(5X).

III. Storage-20℃

3


v201906Da


IV. ProtocolsTwoprotocolsaredescribed:aStandardProtocol,inwhichanextensiontimeof1min/kbisused;andaRapidPCRProtocol,inwhichextensioncanbeconductedat10sec/kbbyusingtwicethequantityofenzyme.PCRreactionmixturescanbepreparedatroomtemperature.However,keepeachofthereactioncomponentsonicewhilepreparingthereactionmixture.

A. Standard Protocol

・ CompositionofthePCRReactionMixtureReagent Volume/Amount Finalconc.5XPrimeSTARGXLBuffer 10μl 1XdNTPMixture(2.5mMeach) 4μl 200μMeachprimer1 10-15pmol 0.2-0.3μM＊primer2 10-15pmol 0.2-0.3μM＊Template RefertoSectionV.3.OptimizationofParametersPrimeSTARGXLDNAPolymerase 1μl 1.25U/50μlSterilepurifiedwater to50μl＊ Whenamplifyingproducts≧10kbinlength,useprimersatafinalconcentrationof0.2μMeach.

・ PCRConditions[For≦10kbproducts]98℃ 10sec55or60℃＊1 15sec 30cycles[3-stepPCR]68℃＊2 1min/kb-or-98℃ 10sec 30cycles[2-stepPCR]68℃＊2 1min/kb

＊1 WhentheTmvalue(calculatedbythefollowingformula＊)isgreaterthan55℃,settheannealingtemperatureto60℃.WhentheTmvalueis55℃orless,settheannealingtemperatureto55℃.＊Tmvaluecalculationmethod:Tm(℃ )=[(thenumberofAandT)x2]+[(thenumberofGandC)x4]-5

＊2 Forboth2-stepand3-stepPCR,settheextensiontemperatureto68℃.

[For10to30kbproducts]98℃ 10 sec 30cycles68℃ 10 min

[For≧30kbproducts]98℃ 10 sec 30cycles68℃ 15 min

◆ SelectingPCRConditions• Foramplificationofproducts≦10kbinlength,try3-stepPCRfirst.• WhenusingGC-richtemplatesoramplifyingproducts≧10kbinlength,2-stepPCRisrecommended.• Ifamplifiedproductsarenotobtained,orifasmearoranon-specificband(s)isobservedduringelectrophoreticanalysis,refertoSectionVII.Troubleshooting.

4


v201906Da


B. Rapid PCR Protocol・ CompositionofPCRReactionMixture

Reagent Volume/Amount Finalconc.5XPrimeSTARGXLBuffer 10μl 1XdNTPMixture（2.5mMeach） 4μl 200μMeachprimer1 10-15pmol 0.2-0.3μM＊primer2 10-15pmol 0.2-0.3μM＊Template RefertoSectionV.3.OptimizationofParametersPrimeSTARGXLDNAPolymerase 2μl 2.5U/50μlSterilepurifiedwater to50μl＊ Whenamplifyingproducts≧10kbinlength,useprimersatafinalconcentrationof0.2μMeach.

・ PCRConditions[For≦10kbproducts]98℃ 10sec55or60℃＊1 15sec 30cycles[3-stepPCR]68℃＊2 10sec/kb

＊1 WhentheTmvalue(calculatedbythefollowingformula＊)ismorethan55℃,settheannealingtemperatureto60℃.WhentheTmvalueis55℃orless,settheannealingtemperatureto55℃.＊Tmvaluecalculationmethod:Tm(℃ )=[(thenumberofAandT)x2]+[(thenumberofGandC)x4]-5

＊2 For3-stepPCR,settheextensiontemperatureto68℃.

[For10to20kbproducts]98℃68℃

10sec20sec/kb 30cycles[2-stepPCR]

-or-98℃60℃68℃

10sec15sec

10sec/kb30cycles[3-stepPCR]

◆ SelectingPCRConditions• Foramplificationofproducts≦10kbinlength,perform3-stepPCR.2-stepPCRisnotrecommendedforproductsofthissize.• Foramplificationofproducts≧10kbinlength,3-stepPCRisrecommendedwhenashorterreactiontimeisdesired,and2-stepPCRisrecommendedwhenenhancedspecificityisdesired.• ForGC-richtemplates,usetheStandardProtocol.• Ifamplifiedproductsarenotobtained,orifasmearoranon-specificband(s)isobservedinelectrophoreticanalysis,refertoSectionVII.Troubleshooting.

5


v201906Da


V. Optimization of ParametersInordertoobtainthebestPCRresults,itisimportanttooptimizethePrimeSTARGXLDNAPolymerasereactionparameterstofullyutilizetheenzyme'spropertiesandadvantages.

(1)PrimerdesignSelectprimersequencesusingprimerdesignsoftwaresuchasOLIGOPrimerAnalysisSoftware(MolecularBiologyInsights,Inc.).

[For≦10kbproducts]Forgeneralamplification,20-to25-merprimersaresuitable.SelectionofprimerswithaTmvalueof≧55℃(ascalculatedusingtheformulainSectionIV.PCRConditions)orgreaterthan25-merinlengthmayprovideoptimalresults.SeeSectionIV.Protocols.

[For>10kbproducts]Designprimersthatare25-to35-mersandthathaveaTmvalueof≧65℃.AvoidhighGC-contentatthe3'endofeachprimer.

[ForGC-richamplificationproducts]DesignprimerstohaveTmvalues>60℃.

Note:Donotuseinosine-containingprimerswithPrimeSTARGXLDNAPolymerase.

(2)dNTPandMg2+dNTPsarecapableofchelation,andthereforetheconcentrationoffreeMg2+inareactionmixisinverselyrelatedtodNTPconcentration.ThePrimeSTARGXLBufferisformulatedtoresultinafinal(1X)concentrationof1mMMg2+whenthefinal(1X)concentrationofdNTPsis200μMeach.AvoidchangingthedNTPconcentrationasmuchaspossible.DonotusedUTPwithPrimeSTARGXLDNAPolymerase.dUTPwillgreatlyaffectenzymeactivity.

(3)TemplateRecommendedquantitiesoftemplateDNA(assuminga50μlreaction):

(forgeneralconditions) (forlongPCRproducts)HumangenomicDNA 5-500ng 100-500ngE.coli genomicDNA 100pg-200ng 10-200ngPlasmidDNA 10pg-10ng 1-10ngcDNA 25-750ng 250-750ng

Donotusetemplatescontaininguracil,suchasbisulfite-treatedDNA.

6


v201906Da


VI. Electrophoresis, and Cloning of Amplified Products(1)Electrophoresis

TAEBufferisrecommendedforagarosegelelectrophoresisofamplifiedproductsthatareobtainedusingPrimeSTARGXLDNAPolymerase.Note: TBEBuffermayresultinDNAbandpatternsthatareenlargedatthebottomof

thegel.

(2)TerminiofamplifiedproductsMostPCRproductsamplifiedwithPrimeSTARGXLDNAPolymerasehaveblunt-endtermini.Therefore,theycanbecloneddirectlyintoblunt-endvectors.Ifnecessary,phosphorylateamplifiedproductsbeforecloning.UsetheMightyCloningReagentSet(BluntEnd)(Cat.#6027)forcloningintoablunt-endvector.

(3)RestrictionenzymedigestionPriortoperformingrestrictionenzymedigestionofamplifiedPCRproducts,removealltracesofPrimeSTARGXLDNAPolymerasefromthereactionmixturebyphenol/chloroformextractionorbyusingtheNucleoSpinGelandPCRClean-Up(Cat.#740609.50/.250)kit.Forrestrictionenzymesthatproducea3'-overhang,suchasPst I,the3'-overhangmayberemovedbythe3'→5'exonucleaseactivityofPrimeSTARGXLDNAPolymeraseifresidualpolymeraseremainsintherestrictiondigestreaction.

VII. Troubleshooting

Event PossibleCauses Action

Noamplificationorpooramplificationefficiency

PrimerTm RefertoSectionV.(1)OptimizationofParameters-Primerdesign

Annealingtemperature Lowerby2℃pertrialPrimerconcentration Useintherangeof0.3to0.5μM(finalconc.)PCRconditions TrytheRapidPCRProtocolNumberofcycles Use35to40cyclesPurityandquantityoftemplateDNA

UseanappropriateamountoftemplateDNAPurifythetemplateDNA

Electrophoreticanalysisshowsasmearedband(s)orextraband(s)

PrimerTm RefertoSectionV.(1)OptimizationofParameters-Primerdesign

Annealingtemperature

Raiseby2℃pertrialuptoto63℃Try2-stepPCRForaprimerTmvalueof50℃orless,setintherangeof50to55℃

Extensiontime Whenamplificationproductis≦1kb,setto10sec/kb

Primerconcentration Useatafinalconcentrationof0.2μMeachNumberofcycles Use25to30cyclesTemplateDNApurity PurifythetemplateDNA

7


v201906Da


VIII. FeaturesA. Accuracy

ThemutationfrequencyofPrimeSTARGXLDNAPolymerasewasexaminedbyanalysisofsequencingdata.[Method]TenarbitrarilyselectedGC-richregionswereamplifiedwithPrimeSTARGXLDNAPolymeraseorotherDNApolymerasesusingThermusthermophilus HB8genomicDNAasatemplate.EachPCRproduct(approx.500bpeach)wasclonedintoasuitableplasmid.Multiplecloneswereselectedforeachamplificationproductandweresubjectedtosequenceanalysis.

[Result]SequenceanalysisofDNAfragmentsamplifiedusingPrimeSTARGXLDNAPolymeraseshowedonly30mismatchedbasesper486,923totalbases.ThisishigherfidelitythanPfuDNAPolymerase.

B. Length of amplification productsPrimeSTARGXLDNAPolymeraseenablestheamplificationoflongDNAfragmentsthatcannotbeobtainedusingothercommerciallyavailablehigh-fidelityPCRenzymes.Asshownbelow,amplificationwasconfirmedforfragmentsupto30kbinlengthusinghumangenomicDNAasthetemplate,upto40kbinlengthusingλDNAasthetemplate,andupto13.5kbinlengthusingcDNAasthetemplate.

0.045(%)0.040.0150.010.0050

rTaqDNAPolymerase

PfuDNAPolymerase

PrimeSTARGXLPolymerase

PrimeSTARHSPolymerase

PrimeSTARMaxPolymerase (12/542,580)

(15/484,429)

(30/486,923)

(13/199,186)

(64/159,994)

8


v201906Da


1. 0.5kb2. 1kb3. 2kb4. 4kb5. 8kb6. 15kb7. 20kb8. 30kb9. 40kbM.λ-HindIIIdigest

λDNA(Template:1ng/50μlreaction)

M 1 2 3 4 5 6 7 8 9 M

1. p53 0.5kb 2. DCLRE1A 1kb 3. DCLRE1A 2kb 4. DCLRE1A 4kb 5. β-globin 8.5kb 6. β-globin 15kb 7. β-globin 20kb 8. β-globin 24kb 9. β-globin 27kb10. β-globin 30kb M. λ-HindIIIdigest

HumangenomicDNA(Template:100ng/50μlreaction)

M 1 2 3 4 5 6 7 8 9 10 M

1.Dystrophin 1kb2.Dystrophin 2kb3.CCND2 2.8kb4.TFR 4kb5.Dystrophin 6kb6.Dystrophin 8kb7.Dystrophin 12kb8.Dystrophin 13.5kbM.λ-HindIIIdigest

(Template:equivalentof250ngtotalRNA/50μlreaction)

M 1 2 3 4 5 6 7 8 M

cDNA

C. Amplification of GC-rich targetsPrimeSTARGXLDNAPolymeraseallowsforhighlyspecificamplificationofGC-richtemplatesthatareotherwisechallenging.Excellentresultsareachievedwithoutspecialbuffersorreactionconditions.TheperformanceofPrimeSTARGXLDNAPolymeraseincomparisontoothercommerciallyavailablehigh-fidelityDNApolymerasesandpolymerasesoptimizedforGC-richtemplatesisshownbelow.Reactionswereperformedaccordingtotheprotocolsspecifiedbyeachmanufacturer.

M 1 2 3 4 M 1 2 3 4 M M 1 2 3 4 M 1 2 3 4 M 1 2 3 4 MM 1 2 3 4 M

PrimeSTAR GXLPolymerase

Company A's enzyme

Company B's enzyme

Company B's enzyme 2

Company C's enzyme

Company D's enzyme

Template:HumangenomicDNA(100ng/50μlreaction)1. APOEgene 746bp(GC-content74%)2. TGFβ1gene 2,005bp(GC-content69%)

Template:T.thermophilus HB8genomicDNA (10ng/50μlreaction)3. 2,029bp (GC-content74%)4. 4,988bp (GC-content74%)

M:pHYMarker

CompanyB'senzyme2:OptimizedforGC-richtemplates

CompanyD'senzyme:IncludesbuffersoptimizedforGC-richtemplates

9


v201906Da


D. Sensitivity and range of template quantityConventionalhigh-fidelityPCRenzymesarerelativelyeasilyaffectedbyexcessnucleicacidinthereactionmixture,andfrequentlydonotreadilyamplifycDNAtemplates.Incontrast,PrimeSTARGXLDNAPolymeraseshowsexcellentactivityoverawiderangeoftemplatequantities,andtherefore,iswell-suitedtoamplificationofcDNAtemplates.

(1) UsingcDNAtemplatesobtainedbyreversetranscriptionofvariousquantitiesoftotalRNApreparedfromHL-60cells,thetransferrinreceptor(TFR)gene(4kb)wasamplifiedusingeachenzymeinthePrimeSTARseries.Sensitivityandtherangeoftemplatequantitywerecompared.

cDNAtemplatequantity(equivalenttototalRNAamounts/50μlreaction): 1. 25pg 7. 750ng 2. 250pg 8. 1μg 3. 2.5ng 9. 1.5μg 4. 25ng 10. 2μg 5. 250ng M. λ-HindIIIdigest 6．500ng

PrimeSTARGXLDNAPolymerasedemonstratedgoodamplificationoverawiderangeoftemplatecDNAquantity,aswellasexcellentsensitivity.

MM 1 2 3 4 5 6 7 8 9 10 M M 1 2 3 4 5 6 7 8 9 10 M 1 2 3 4 5 6 7 8 9 10PrimeSTAR GXL Polymerase PrimeSTAR HS Polymerase PrimeSTAR Max Polymerase

10


v201906Da


(2) UsingvariousquantitiesofhumangenomicDNAasatemplate,theamplificationefficiencyofPrimeSTARGXLDNAPolymerasewascomparedtotheefficienciesofothercommerciallyavailablehigh-fidelityPCRenzymesandTaqDNAPolymerase.Reactionswereperformedaccordingtotheprotocolsspecifiedbyeachmanufacturer.

Templatequantity(per50μlreaction):1.Notemplate2． 100pg3． 1ng4． 10ng5． 100ng6． 200ng7． 500ngM．λ-HindIIIdigest

Company A's enzyme

PrimeSTAR GXLPolymerase

M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 M

Company C's enzyme

Company B's enzyme

M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 M

Company E's enzyme

Taq DNA Polymerase

M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 M

Template:HumangenomicDNATarget:DCLRE1Agene(2kb)

PrimeSTARGXLDNAPolymerasedemonstratedsuperiorsensitivityandamplificationefficiencyincomparisontoothercommerciallyavailablehigh-fidelityPCRenzymesandTaqDNAPolymerase.HighactivitywasobservedforPrimeSTARGXLDNAPolymeraseeveninthepresenceofexcesstemplateDNAthatsuppressedtheactivityofhigh-fidelityPCRenzymesfromothercompanies.

IX. Related Products

PrimeSTAR®MaxDNAPolymerase(Cat.#R045A/B)PrimeSTAR®HSDNAPolymerase(Cat.#R010A/B)PrimeSTAR®HS(Premix)(Cat.#R040A)TaKaRaPCRThermalCyclerDice™Gradient(Cat.#TP600)MightyCloningReagentSet(BluntEnd)(Cat.#6027)NucleoSpinGelandPCRClean-Up(Cat.#740609.50/.250)*AgaroseL03「TAKARA」(Cat.#5003)PrimeGel™AgarosePCR-Sieve(Cat.#5810A)

＊Notavailableinallgeographiclocations.Checkforavailabilityinyourarea.

11


v201906Da


PrimeSTARisaregisteredtrademarkofTakaraBioInc.ThermalCyclerDiceandPrimeGelaretrademarksofTakaraBioInc.

NOTE : Thisproductisforresearchuseonly.Itisnotintendedforuseintherapeuticordiagnosticproceduresforhumansoranimals.Also,donotusethisproductasfood,cosmetic,orhouseholditem,etc.Takaraproductsmaynotberesoldortransferred,modifiedforresaleortransfer,orusedtomanufacturecommercialproductswithoutwrittenapprovalfromTakaraBioInc.Ifyourequirelicensesforotheruse,pleasecontactusbyphoneat+81775656972orfromourwebsiteatwww.takara-bio.com.Youruseofthisproductisalsosubjecttocompliancewithanyapplicablelicensingrequirementsdescribedontheproductwebpage.Itisyourresponsibilitytoreview,understandandadheretoanyrestrictionsimposedbysuchstatements.Alltrademarksarethepropertyoftheirrespectiveowners.Certaintrademarksmaynotberegisteredinalljurisdictions.

12


v201906Da


1sjnf453 (9- %/1pmznfsbtf · (1) primer design select primer sequences using primer design software...

Documents