KOD高保真酶说明书

(81)-6-6348-3888Tel(86)[e-mail]SE.1InstructionmanualKOD-Plus-0902F0934KKOD-Plus-KOD-201200U200reactionsStoreat-20°CContents[1]Introduction[2]Components[3]Qualitytesting[4]Primerdesign[5]CloningofPCRproducts[6]Protocol1.Standardreactionsetup2.Cyclingconditions[7]Examples[8]Troubleshooting[9]References[10]RelatedproductsCAUSIONAllreagentsinthiskitareintendedforresearchpurposes.Donotusefordiagnosisorclinicalpurposes.Pleaseobservegenerallaboratoryprecautionandutilizesafetywhileusingthiskit.(81)-6-6348-3888Tel(86)[e-mail]SE.1[1]Introduction[2]Components[3]QualityTestingDescriptionKOD-Plus-isbasedonDNApolymerasefromthehyperthermophilicArchaeonThermococcuskodakaraensisKOD11)2).KOD-Plus-exhibitsexcellenthighPCRfidelityandefficiency.TheenzymesolutionofKOD-Plus-containstwotypesofanti-KODDNApolymeraseantibodiesthatinhibitpolymeraseand3’J5’exonucleaseactivity,thusallowingforHotStartPCR3).KOD-Plus-generatesblunt-endPCRproducts,dueto3’J5’exonuclease(proof-reading)activity.Features-HotStarttechnology,usinganti-KODDNApolymeraseantibodies,resultsinhighlyefficientamplification(seeExample1).-KOD-Plus-enablesthefollowingamplifications(maximum):21kbfromlambdaphageDNA,12kbfromhumangenomicDNA,and7kbfromcDNA.-KODDNApolymerasehasstrong3’J5’exonuclease(proof-reading)activity.ThePCRerrorratioofKOD-Plus-isapprox.80timeslessthanTaqDNApolymerase.Table.1PCRfidelitycomparisonofeachPCRenzyme.*PCRfidelitywasbasedonthemutationfrequencyofPCRproductsusingapositive-selectionbaseassaywiththerpsLgene4).Thisreagentincludesthefollowingcomponentsfor200reactions:KOD-Plus-(1.0U/μl)*200μl×110×BufferforKOD-Plus-1.0ml×125mMMgSO41.0ml×12mMdNTPs1.0ml×1*Theenzymesolutioncontainsanti-KODDNApolymeraseantibodiesthatneutralizepolymeraseand3’J5’exonucleaseactivity.Qualitycheckcanbeperformedbyamplifyingtheβ-globingene(3.6Kb)andp53gene(4.0Kb).TotalMutantKOD-Plus-10,610100.09HighfidelityPCRenzyme(Acompany)10,900680.62PfubasedDNApolymerase6,520761.17TaqDNApolymerase10,5607807.39Mutationfrequency(%)Colonynumber(81)-6-6348-3888Tel(86)[e-mail]SE.2[4]PrimerDesign[5]CloningofPCRproducts[6]Protocol-Primersshouldbe22-34baseswithameltingtemperature(Tm)over60°C.Foramplificationofalongtarget,25-34baseswithhighTmvalues(≥65°C)arerecommended.PCRprimersshouldbedesignedaccordingtothegeneralguidelines.-KOD-Plus-generatesblunt-endPCRproducts,dueto3’J5’exonuclease(proof-reading)activity.Therefore,theproductcanbeclonedaccordingtoablunt-endcloningmethod.-PCRproductsofKOD-Plus-shouldbepurifiedpriortorestrictionenzymetreatments.The3’J5’exonucleaseactivityofKODDNApolymeraseremainsafterthePCRcycles.1.StandardreactionsetupThefollowingprocedureisdesignedforusewiththecomponentsprovidedinthiskit.Beforepreparingmixture,allcomponentsshouldbecompletelythawed,exceptfortheenzymesolution.*DonotusedNTPsfromotherkitsorcompanies.Notes:-ForPCRreactions,thin-walltubesarerecommended.Atotalreactionvolumeof50μlisalsorecommended.-TheadditionofDMSO(finalconc.2-5%)mightbeeffectiveforamplificationofGC-richtargets.DecreasedPCRfidelityhasbeenconfirmedtonottakeplacewithDMSO.-ContaminatedRNA(usedforcDNA)orgenomicDNAinhibitsthePCRreactionbychelatingMg2+.PCRshouldbeperformedusingtemplateDNAcontaining100ngRNAcomponent.ComponentVolumeFinalConcentration10xBufferforKOD-Plus-5μl1x2mMdNTPs*5μl0.2mMeach25mMMgSO42μl1.0mM10pmol/μlPrimer#11.5μl0.3μM10pmol/μlPrimer#21.5μl0.3μMTemplateDNAXμlGenomicDNA10-200ng/50μlPlasmidDNA1-50ng/50μlcDNA≤100ng（RNAequiv.)/50μlPCRgradewaterYμlKOD-Plus-(1.0U/μl)1μl1.0U/50μlTotalreactionvolume50μl(81)-6-6348-3888Tel(86)[e-mail]:IftheTmvalueoftheprimerisunder73°C,the3-stepcycleisrecommended.*Tmvalueoftheprimerminus5°C-10°CNotes:-Extensiontimeshouldbesetto1minper1kboftargetlength.2-stepcyclePre-denaturation:94°C,2min.Denaturation:94°C,15sec.Extension:68°C,1min./kb3-stepcyclePre-denaturation:94°C,2min.Denaturation:94°C,15sec.Annealing:Tm-[5-10]oC*,30sec.Extension:68°C,1min./kb25-35cycles25-35cycles(81)-6-6348-3888Tel(86)[e-mail]SE.4[7]ExamplesExample1．EffectofHotStartPCRonthegenerationofprimerdimers.Example2.EffectofadditionofDMSOforGC-richtargets.Template:HumangenomicDNATarget:TGF-βgene(