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Pfu DNA Polymerase | Taq DNA Polimerase High Fidelity Cellco
Pfu DNA Polymerase
Ficha técnica
| Código | Pfu DNA Polymerase |
| Categoria | PCR Convencional |
| Marca | Cellco |
Descrição Geral
Pfu DNA Polymerase | Taq DNA Polimerase High Fidelity - Cellco
DNA polimerase com atividade proofreading, elevada acurácia
Pyrococcus furiosus, recombinante, E. coli
For in vitro use only!
Definição de unidade: One unit is defined as the amount of the enzyme required to catalyze the incorporation of 10 nmoles of dNTPs into an acidinsoluble form in 30 minutes at 70 °C.
Envio: Shipped on blue ice
Condições de armazenamento: Store at -20 °C
Avoid freeze/thaw cycles
Validade: 12 months
Concentração: 2,5 units/μL
Kit contents:
Pfu (blue cap)
2,5 units/µl Pfu in 20 mM Tris-HCl pH 8.0 (25°C), KCl, EDTA, DTT, 50% (v/v) Glycerol and stabilizers.
Pfu Reaction Buffer complete (red cap) - 10x
Tris-HCl pH 8,8 (25°C), KCl and 25 mM MgCl2.
Descrição:
Pfu Pol is a thermostable proofreading enzyme specially designed for highly accurate and efficient amplification it replicates into dsDNA at 72 °C . It catalyzes the polymerization of nucleotide in the 5' → 3' direction in the presence of magnesium and exhibits a 3' → 5' exonuclease (proofreading) activity. This features results in a greatly increased fidelity of DNA synthesis compared to Taq polymerase. The enzyme is highly purified and free of bacterial DNA. The amplification results in blunt-ended fragments.
Fidelidade da Enzima:
Pfu Pol is characterized by a 6x higher fidelity compared to Taq DNA Polymerase.
PCR Reaction Setup:
The PCR procedure below shows appropriate volumes for a single 50-μL reaction. For multiple reactions, prepare a master mix of components common to all and then dispense appropriate volumes into each PCR reaction tube prior to adding template DNA and primers. Thaw, mix, and briefly centrifuge each component before use.
Add the following components to a microcentrifuge tube:
1. Prepare PCR master mix
Note: Consider the volumes for all components listed next steps to determine the correct amount of water required to reach your final reaction volume.
| Component | 50 μL rxn | Final conc. |
| Water, grade PCR | to 50 μl | - |
| 10x Reaction Buffer | 5 μl | 1x |
| dNTP (Mix 10 mM) | 1 μl | 200 μM |
| Pfu Pol (2,5U/ µl) | 0,5 μl | 1,25 U/reaction |
Mix and briefly centrifuge the components.
2. Add template DNA and primers
| Component | 50 μl rxn | final conc. |
| Forward primer (10 μM) | 0,5 – 2,5 μl | 0,1 – 0,5 μM |
| Reverse primer (10 μM) | 0,5 – 2,5 μl | 0,1 – 0,5 μM |
| Template DNA | x μl | 1 pg – 1 μg** |
**genomic DNA: 1 ng – 1 μg; plasmidial DNA: 1 pg – 1 ng
Cap each tube, mix, and briefly centrifuge the content.
3. Incubate reactions in a thermal cycler
Recommended cycling conditions:
| Initial denaturation | 98 °C | 30 sec | 1x |
| Denaturation | 98 °C | 5 - 10 sec | 25-35 cycles |
| Annealing** | 45 - 70 °C | 15 - 30 sec | 25-35 cycles |
| Elongation*** | 72 °C | 1 min/kb | 25-35 cycles |
| Final extension (optional) | 72 °C | 1 – 2 min/kb | 1x |
| Hold | 4 – 8 °C | ∞ | 1x |
**The annealing temperature depends on the melting temperature of the primers used
***The elongation time depends on the length of the fragments to be amplified. A time of 1 min/kb is recommended.
For optimal specificity and amplification an individual optimization of the recommended parameters may be necessary for each new template DNA and/or primer pair.
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