Cat. # R110A

Product Manual

TaKaRa EpiTaq™ HS (for bisulfite-treated DNA)

For Research Use

v201910Da

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

TaKaRa EpiTaq™ HS (for bisulfite-treated DNA)Cat. #R110A

v201910Da

Table of Contents

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

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

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

IV. CompositionofPCRMixture........................................................................ 3

V. PCRConditions.................................................................................................. 4

VI. ElectrophoresisofAmplificationProducts............................................. 4

VII. PCRProducts....................................................................................................... 4

VIII. PrimerDesign..................................................................................................... 5

IX. ExperimentalExamples.................................................................................. 6

X. Troubleshooting................................................................................................ 7

XI. RelatedProducts............................................................................................... 8

TaKaRa EpiTaq™ HS (for bisulfite-treated DNA)Cat. #R110A

v201910Da

3URL:http://www.takara-bio.com

I. DescriptionTaKaRaEpiTaqHS(forbisulfite-treatedDNA)isaDNApolymeraseoptimizedforPCRamplificationsusingbisulfite-treatedDNAcontaininguracilastemplate.PCRreactionswithbisulfite-treatedDNAoftenposeparticulartechnicalchallengesforconventionalenzymes.TaKaRaEpiTaqHSandthereagentsinthiskit,however,allowadjustmentofmagnesiumanddNTPconcentrationstoachieveoptimalamplificationefficienciesandspecificities,facilitatingexcellentamplificationsevenfortargetsthatpreviouslyweredifficulttoamplify.TaKaRaEpiTaqHSisalsoahot-startPCRenzymewithananti-Taqantibody.Priortohigh-temperaturedenaturation,theanti-Taqantibodysuppressespolymeraseactivity,therebypreventingnon-specificamplificationsasaresultofmisprimingorprimerdimerformationbeforestartingPCR.ThisallowstheuseofconventionalPCRconditionswithoutrequiringaspecialdenaturationstep.

II. Components1. TaKaRaEpiTaqHS（5U/μl） 250U2. 10XEpiTaqPCRBuffer(Mg2+free) 1ml3. dNTPMixture（2.5mMeach） 1.2ml4. 25mMMgCl2 1.2ml

III. Storage -20℃

IV. Composition of PCR Mixture

Reagent Volume/Amount FinalCon.TaKaRaEpiTaqHS（5U/μl） 0.25μl 1.25U/50μl10XEpiTaqPCRBuffer(Mg2+free) 5μl 1X25mMMgCl2 5μl 2.5mMdNTPMixture（2.5mMeach） 6μl 0.3mMTemplate <100ngPrimer1 20pmol 0.4μMPrimer2 20pmol 0.4μMSterilepurifiedwater to50μl

Forafirsttrial,usethereactionmixturedescribedabove.Ifthisreactionmixturefailstoresultindesiredlevelsofproductamplificationorresultsintheappearanceofextrabands,adjustingtheconcentrationofMgCl2,dNTPmixture,orprimermayimproveresults.Fordetails,seesectionX.Troubleshooting.PCRreactionmixturescanbepreparedatroomtemperature.Eachcomponent,however,shouldbekeptonice.

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

TaKaRa EpiTaq™ HS (for bisulfite-treated DNA)Cat. #R110A

v201910Da

V. PCR Conditions

98℃55℃72℃

10sec＊1 30sec 30sec(~500bp)or1min(500bp~1kb)＊2

30-40cycles

＊ 1 SelectdenaturationconditionscompatiblewiththePCRthermalcyclerandtubesusedforthereaction.Ingeneral,treatat98℃for5-10secorat94℃for20-30sec.

＊ 2 Setextensiontimeaccordingtotheamplificationsize.Ingeneral,use30secforanamplificationproductsofupto500bp,and1minforamplificationproductsrangingfrom500bpto1kb.Asageneralguideline,use1minper1kbwhentheproductsizeexceeds1kb.

AstemplateDNAwasdamagedbybisulfitetreatment,amplificationefficiencyoflargerproductsmaybedecrease.

VI. Electrophoresis of Amplification Products

Add6XLoadingBuffer(suppliedwithTaKaRaDNAmolecularweightmarkers)toanaliquotofthePCRreactionmixture(5-10μl)inaratioof1:5byvolume,andanalyzebyagarosegelelectrophoresis.Afterelectrophoresis,stainthegelusing1.0μg/mlofethidiumbromidefor20-30min.VisualizeDNAbandsunderUVirradiation.

VII. PCR Products

ThemajorityofPCRproductsamplifiedwithTaKaRaEpiTaqHShaveasingleAnucleotideaddedat3’termini,allowingthePCRproductstobecloneddirectlyintoT-vectorssuchaspMD20(Cat.#3270)orpMD19(Simple)(Cat.#3271).ThesePCRproductsmayalsobeusedforblunt-endcloningafterbluntingandphosphorylation.

TaKaRa EpiTaq™ HS (for bisulfite-treated DNA)Cat. #R110A

v201910Da

5URL:http://www.takara-bio.com

VIII. Primer DesignTheprimerdesigntoolsbelowarerecommendedtodesignprimersforPCRamplifica-tionusingbisulfite-treatedDNAastemplate.Thedesigntoolsareavailableonlinefreeofcharge.(Forspecificoperatingprocedures,pleaserefertoeachwebapplication.)Toensuresuccessfulamplificationwithbisulfite-treatedDNAastemplate,anamplificationproductlengthshouldbelessthan300bpamplificationsofuptoapproximately1kbhavebeenperformedusingTakaraEpiTaqHS.(SeesectionIX.ExperimentalExamples.)

BiSearchhttp://bisearch.enzim.hu/?m=msp

MethPrimerhttp://www.urogene.org/methprimer/index1.html

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

TaKaRa EpiTaq™ HS (for bisulfite-treated DNA)Cat. #R110A

v201910Da

IX. Experimental Example

PCRamplificationwasperformedwithbisulfite-treatedHeLagenomicDNAastemplate.

[Method] Bisulfitetreatment : MethylEasy™XceedRapidDNABisulfiteModificationKit（Cat.

#ME002） Target : CpGislandregionupstreamofCDH1,MLH1,orBRCA1genes Amplificationsize : 153bp(CDH1),297bp(CDH1),136bp(MLH1),292bp(MLH1),

613bp(BRCA1),1,017bp(BRCA1)

Reagent Volume FinalCon.Bisulfite-treatedHeLagenomicDNA(50ng/μl)10XEpiTaqPCRBuffer(Mg2+free)25mMMgCl2dNTPMixture（2.5mMeach）SensePrimer（20μM）AntisensePrimer（20μM）TaKaRaEpiTaqHS（5U/μl）Sterilepurifiedwater

2μl5μl5μl6μl1μl1μl

0.25μlto50μl

100ng/50μl1X

2.5mM0.3mM0.4μM0.4μM

1.25U/50μl

98℃55℃72℃

10sec30sec[30secforproduct

TaKaRa EpiTaq™ HS (for bisulfite-treated DNA)Cat. #R110A

v201910Da

7URL:http://www.takara-bio.com

X. Troubleshooting

Event Possiblecauses ActionNoorpooramplificationefficiency

Mg2+concentration RaiseMg2+concentration;try2.5-3mM.(SeeFigure2)

dNTPconcentration LowerdNTPconcentration;try0.2-0.3mM

Annealingtemperature Tryloweringtemperaturefrom55℃.Lowerby2℃pertrial.

Extensiontime Increasetheextensiontimefrom30secto1minorfrom1to2min.

Primerconcentration Raisetheprimerconcentration;try0.4-1μM.

TemplatefragmentationoccurredduringbisulfitetreatmentofDNA

Re-preparebisulfite-treatedtemplateDNA.

Smearedband(s)orextraband(s)

Mg2+concentration LowerMg2+concentration;try2-2.5mM

dNTPconcentration RaisedNTPconcentration;try0.3-0.4mM.

Annealingtemperature Tryraisingtemperaturefrom55℃.Raiseby2℃pertrial.

Primerconcentration Lowertheprimerconcentration;try0.2-0.4μM.

＊ LoweringMg2+concentrationincreasesspecificity,whileraisingMg2+concentrationimprovesamplificationefficiencyandextension.RaisingdNTPconcentrationincreasesspecificity,whileloweringdNTPconcentrationimprovesamplificationefficiencyandextension.

321M M 321M M 321M M

Mg2+:2.5mM Mg2+:3mMMg2+:2.75mM

Figure2.ImprovementswithMg2+concentration

Lane : Size: Gene: 1 : 316bp（CDKN2） 2 : 607bp（MLH1） 3 : 877bp（MLH1） M : 100bpDNALadder

Agarose: AgaroseL03「TAKARA」Gelcon. : 2% Stain : Ethidiumbromide

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

TaKaRa EpiTaq™ HS (for bisulfite-treated DNA)Cat. #R110A

v201910Da

XI. Related ProductsMethylEasy™XceedRapidDNABisulphiteModificationKit(Cat.#ME002)AgaroseL03「TAKARA」(Cat.#5003/5003B)T-VectorpMD20(Cat.#3270)T-VectorpMD19(Simple)(Cat.#3271)

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

EpiTaqisatrademarkofTakaraBioInc.MethyEasyisatrademarkofHumanGeneticSignaturesPty.Ltd.