MCLAB/TaqKlenow/TT200/5,000 Units

Description:
Taq-KlenowismodifiedfromfulllengthTaq-KlenowbytruncatingitsN-terminus,withamolecularweightof61kDa.ComparedwithregularTaq-Klenow,thistruncatedversionisdeficientin5'->3'exonucleaseactivity,butismorethermostableandhashigherfidelityinPCRamplification.

Application:
-PCR(ordinaryandhigh-throughput)
-PrimerExtension
-MicroarrayAnalysis
-Denaturinghighperformanceliquidchromatography(DHPLC)

Source:
ArecombinantE.colistraincarryingtheTaq-KlenowgenefromthethermophilicorganismThermusAquaticusYT-1.

Suppliedwith:
10xTaqPCRBuffer(nodNTP)

Suppliedin:
20mMTris-HCl
100mMNaCl
1.0mMDTT
0.1mMEDTA
Stabilizer
50%Glycerol
pH7.5@25°C

UnitDefinition:
1unitisdefinedastheamountofenzymethatwillincorporate10nmolofdNTPsintoacid-insolublematerialin30minutesat75°C.

PCRGuidelines:
Taq-KlenowistheoriginalandmostcommonlyusedPCRenzyme.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)andlowermayofferincreasesinfidelity.TaqDSC2.0DNAPolymerasecanalsoincorporateandreadthroughdeoxyUridineandInosine,twoanalogsusedincertainapplications.

5.TaqDSC2.0Polymerase:1unit/50µLreaction(20U/mL)istypical,thoughadditionalenzymemaybeaddedtostimulateyields.TaqDSC2.0DNAPolymeraseextendsaDNAtemplateatapproximately1-2000nucleotides/minute,soitisrecommendedthat30-60secondsofextensiontimeshouldbeprovidedperkb,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 (nodNTP)
0.2µl5U/µlTaqDSC2.0DNAPolymerase
19.8µlTypeIWater

RecommendedStorageCondition:-20°C