Catalogue number
ENZ-265
Synonyms
DNA polymerase, EC 2.7.7.7, Pfu polymerase, Pfu-DNA Polymerase.
Introduction
Pfu DNA polymeraseenzyme is found in the hyperthermophilic archaeonPyrococcus furiosus, where it functions in vivoto replicate the organism's DNA. In vitro, Pfu is used to swiftly amplify DNAin the Polymerase Chain Reaction, where the enzyme serves the central function of copying a new strand of DNA during each extension step. Pfu DNA polymerase has superior thermostability and 'proofreading' properties compared to other thermostable polymerases. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease proof reading activity, meaning that it works its way along the DNA from the 5' endto the 3' endand corrects nucleotidemisin corporation errors. Pfu DNA polymerase-generated PCRfragments will have fewer errors than Taq-generated PCR inserts. As a result, Pfu is more commonly used for molecular cloning of PCR fragments than the historically popular Taq. Pfu DNA polymerase is superior for techniques that require high-fidelity DNA synthesis, but can also be used in conjunction with Taq polymerase to obtain the fidelity of Pfu with the speed of Taq polymerase activity.
Description
Pfu DNA Polymerase is a thermo-stable enzyme having a Mw of about 90kDa.
Pfu DNA Polymerase is derived from E. coli that and cloned from Pyrococcus furiosus strain Vc1 DSM3638. Pfu DNA Polymerase replicates DNA at 75°C, catalyzing the polymerization of nucleotides into duplex DNA in the 5′ to 3′ direction in the existence of magnesium. Pfu DNA Polymerase possesses 3′ to 5′ exonuclease (proofreading) activity. Base misinsertions that take place during polymerization are swiftly removed by the proofreading activity of the polymerase. Therefore, Pfu DNA Polymerase is suggested for use in PCR and primer extension reactions that require high-fidelity synthesis. Pfu DNA Polymerase-generated PCR fragments are blunt-ended.
Source
Escherichia Coli.
Physical Appearance
Sterile liquid formulation.
Formulation
50mM Tris-HCl, pH 8.2, 1mM DTT, 0.1mM EDTA, 0.05% CHAPS and 50% glycerol.
Stability
Pfu DNA Polymerase although stable at 10°C for 5 days, should be stored below -18°C.
Please prevent freeze-thaw cycles.
Unit Definition
1U of enzyme catalyzes the incorporation of 10nmol of dNTP into acid-insoluble product in 30 minutes at 75°C.
10X Buffer with MgSO4
200mM Tris-HCl (pH 8.8 at 25°C), 100mM (NH4)2SO4, 100mM KCl, 1% Triton X-100, 1mg/ml BSA, 20mM MgSO4.
PCR Protocol
Add the following components to amplify 1kb DNA template:
0.2μl Pfu-DNA Polymerase.
4μl 2.5mM dNTPs.
5μl 10x buffer with MgSO4.
1μl Primers mix (10μM each).
1μl Template.
38μl ddH2O.
Amplify using the following cycling parameters:
Heat Soak: 1 cycle at 94°C/4 min.
Denaturation: 30 cycles at 94°C/30 sec.
Annealing: 30 cycles at 55°C /30 sec.
Extension: 30 cycles at 72°C /90 sec.
Final: 1 cycle at 72°C /5 min.
Applications
1. Ideal for high-fidelity amplification.
2. 3'-5' exonuclease activity provides a low error rate.
3. One of the most thermostable DNA polymerases known.
4. Lack of extendase activity means no unwanted 3’ overhangs.
5. Optimal for blunt-end PCR cloning.
6. Optimum temperature near 75°C.
7. 95% active after 1-hour incubation at 98°C.
Safety Data Sheet
Usage
Prospec's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.
