Description: TaqDNAPolymerase(regular)isathermostableenzymewithahighlyprocessive5'->3'polymeraseactivityand5'->3'exonucleaseactivity.TheenzymeispurifiedfromE.coliandconsistsofasinglepolypeptidewithamolecularweightof94kDa.TaqDNApolymerasesynthesizesDNAfromsingle-strandedtemplatesinthepresenceofdNTPsandaprimer.
Application: -PCR(ordinaryandhigh-throughput) -PrimerExtension -MicroarrayAnalysis -Denaturinghighperformanceliquidchromatography(DHPLC)
Source: ArecombinantE.colistraincarryingtheTaqDNApolymerasegenefromthethermophilicorganismThermusAquaticusYT-1.
SuppliedWith: 10xTaqPCRBuffer(nodNTP)
SuppliedIn: 20mMTris-HCl 100mMNaCl 1.0mMDTT 0.1mMEDTA 50%Glycerol pH7.5@25°C
UnitDefinition: 1unitisdefinedastheamountofenzymethatwillincorporate10nmolofdNTPsintoacid-insolublematerialin30minutesat75°C.
PCRGuidelines: TaqDNAPolymeraseistheoriginalandmostcommonlyusedPCRenzyme.Taqexcelsatamplifyingshorter(<5kb)sequencesfromlow-complexitytemplatesourcesandproducesrobustyieldswithlittleornooptimizationofreactionconditions.ConsiderthefollowingguidelineswhendesigningPCRstrategiesusingTaqDSC2.0DNAPolymerase.
1.DNATemplate:AlthoughextensivepurificationofPCRtemplatesistypicallynotnecessary,careshouldbetakenwithcrudeorpartiallypurifiedDNAsourcesashandlingandchemicalagentscanadverselyaffectthePCRprocess.Exposuretoshort-waveUVlightorotherDNAdamagingagentsshouldbeavoided,asshouldhighionicstrength,detergentssuchasSDS,loADIngdyesandphenol.InordertopreventcontaminationfrompreviousPCRreactions,considersettingupreactionsinapositive-pressurehoodandwithaerosolbarrierpipettips.Inatypical25cyclePCR,104copiesoftargetsequencewillyieldreproducIBLeamplificationproduct.Thiscorrespondstoroughly0.1-1ng/ml(finalconcentration)ofplasmidDNA,and1-10µg/mlofgenomicDNA.TheuseoflowerDNAconcentrationstypicallyproduceslessnon-specificproduct,whilehigherconcentrationscanallowforfewercyclesandlowermutationrates.
2.PrimerDesign:Ideally,oligonucleotideprimersare15-30basesinlength,nearly50%G+C,andhaveequal(+/-3°C)annealingtemperatures.Theuseofsoftwaretodetectself-complementaryorhairpin-proneregionsisadvisedandisofferedasaservicebysomesynthesisproviders.Notethatalthoughthe5'-terminusoftheprimermaycontainuntemplatedsequence,the3'endmustmatchperfectly.Typicaloligonucleotideconcentrationinthereactionis0.1-0.5µM.
3.Magnesium:MagnesiumisacriticalcomponentofthePCRreactionthoughitsconcentrationcanbemodulatedtopromotevariouseffects.Generally,1.5-2.0mMMg2+istargeted,buthigherconcentrations(upto5mM)maybeusedtostimulatetheyieldofreactionsattheexpenseoffidelity.Theconverseisalsotrue-lowermagnesiumconcentrationswillpromotehigher-fidelityproductswithaloweroverallamplificationyield.Notethatcertainreactioncomponents,inparticulartemplateDNAandoligonucleotides,maycontributechelatingagentstothereactionwhichcouldlowertheeffectivemagnesiumconcentrationandstarvethereaction.
4.dNTPs:Generally,afinalconcentrationof100-200µMdNTPsisemployed,thoughhigherconcentrationsmaystimulateyields(particularlywithlongertargets)andlowerconcentrations mayofferincreasesinfidelity.TaqDSC2.0DNAPolymerasecanalsoincorporateandreadthroughdeoxyUridineandInosine,twoanalogsusedincertainapplications.
5.TaqDSC2.0Polymerase:1unit/50µLreaction(20U/mL)istypical,thoughadditionalenzymemaybeaddedtostimulateyields.TaqDSC2.0DNAPolymeraseextendsaDNAtemplateatapproximately1-2000nucleotides/minute,soitisrecommendedthat30-60secondsofextensiontimebeprovidedperkb,percycle.Appropriateextensiontemperaturesrangefrom68-72°C.BecauseTaqDSC2.0DNAPolymeraseexploitsthenaturalaffinityofaDNApolymeraseforaduplexDNAfragmenttopromoteitshot-startfunction,itdoesnotrequireanextensiveinitialdenaturationsteptoactivatethepolymerase.
Typical50µlReaction: Onice,prepareeachofthefollowingmastermixes,combine,andplaceinheated(to94°C)thermalcycler.
2xDNA/OligonucleotideMasterMix: 1.0µl10mMdNTPs 1.0µl10µMForwardPrimer 1.0µl10µMReversePrimer 1.0µl500ng/µlgenomicDNA 21µlTypeIWater
2xEnzyme/BufferMasterMix: 5.0µl10xTaqPCRBuffer 0.2µl5U/µlTaqDSC2.0DNAPolymerase 19.8µlTypeIWater
RecommendedStorageCondition:-20°C | |