Description:
PfuDNAPolymeraseisahighlythermostableDNApolymerasefromthehyperthermophilicarchaeumPyrococcusfuriosus.Theenzymecatalyzesthetemplate-dependentpolymerizationofnucleotidesintoduplexDNAinthe5'->3'direction.PfuDNAPolymerasealsoexhibits3'->5'exonuclease(proofreADIng)activity,thatenablesthepolymerasetocorrectnucleotideincorporationerrors.Ithasno5'->3'exonucleaseactivity.ThemaindifferencebetweenPfuandalternativeenzymesisPfu'ssuperiorthermostABIlityand'proofreading'properties.UnlikeTaqDNApolymerase,PfuDNApolymerasealsopossesses3'->5'exonucleaseproofreadingactivity,resultinginPCRfragmentswithfewererrorsthanTaq-generatedPCRinserts.PfuDNApolymeraseisefficientfortechniquesthatrequirehigh-fidelityDNAsynthesis,butcanalsobeusedinconjunctionwithTaqpolymerasetoobtainthefidelityofPfuwiththespeedofTaqpolymeraseactivity.

Application:
-High-fidelityPCRandprimer-extensionreactions
-GenerationofPCRproductsforcloningandexpression
-PCRcloningandblunt-endamplificationproductgeneration
-RT-PCRforCDNAcloningandexpression
-Site-directedmutagenesis
-Blunt-endPCRcloning

Source:
ThermostableDNApolymerasefromhyperthermophilicarchaeonpyrococcusfuriosus.

UnitDefinition:
Oneunitisdefinedastheamountofenzymerequiredtocatalyzetheincorporationof10nmolofdNTPsintoacidinsolublematerialin30minutesat74°CunderstandardDNApolymeraseassayconditions.

SuppliedWith:
10xPfuReactionBuffer(withdNTPs)
 

10xPfuReactionBuffer(withdNTPs) 
200mMTn3pH8.8
20mMMgSO₄
100mMKCl
100mM(NH₄)₂SO₄
1%Triton
1mg/mlBSA
2mMdNTP

SuppliedIn:
20mMTris-HCl(pH8.0)
40mMNaCl
0.1mMEDTA
1mMDTT
50%(v/v)glycerol

HeatInactivation:
95%inactiveafter1-hourincubationat98°C

RecommendedReactionConditions:
1XPfubuffer,200µMeachdNTP,0.1-0.5µMeachprimer,5unitsPfuDNApolymeraseenzyme,1-100ngplasmidtemplateDNA,or100-250nggenomictemplateDNA.

RecommendedStorageCondition:-20ºC