Pfu-X Polymerase
Proofreading DNA polymerase for highest accuracy
Pyrococcus furiosus, recombinant, E. coli
Catálogo Nº | Apresentação | Preço (R$) | Comprar |
---|---|---|---|
PCR-207S | 100 units | Sob demanda | Adicionar ao Carrinho |
PCR-207L | 500 units | Sob demanda | Adicionar ao Carrinho |
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 nmol of dNTP into an acid-insoluble form in 30 minutes at 74 °C.
Envio: shipped on blue ice
Condições de armazenamento: store at -20 °C
avoid freeze/thaw cycles
Validade: 12 months
Forma: liquid
Concentração: 2.5 units/μl
Descrição:
Pfu-X Polymerase is the ideal choice for applications where the efficient amplification of DNA with highest fidelity is required. The enzyme is a genetically engineered Pfu DNA polymerase, but showing a 2-fold higher accuracy and an increased processivity, resulting in shorter elongation times.
The enzyme catalyzes the polymerization of nucleotides into duplex DNA in 5'→3' direction but does not possess a 5'→3' exonuclease replacement activity. Its inherent 3'→5' exonuclease proofreading activity results in a greatly increased fidelity of DNA synthesis compared to Taq polymerase. Pfu-X Polymerase-generated PCR fragments are blunt-ended. The enzyme is highly purified and free of bacterial DNA.
Fidelity of the enzyme:
Pfu-X Polymerase is characterized by a 50-fold higher fidelity compared to Taq polymerase and a 2-fold higher fidelity compared to standard Pfu polymerase.
ERPfu-X Polymerase = 0.25 x 10-6
The error rate (ER) of a PCR reaction is calculated using the equation ER = MF/(bp x d), where MF is the mutation frequency, bp is the number of base pairs of the fragment and d is the number of doublings
(2d = amount of product / amount of template).
Contente:
Pfu-X Pol (red cap)
2.5 units/μl Pfu-X Polymerase in storage buffer
(50 % Glycerol, 50 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 1 mM DTT 0.1 % Tween 20, 0.1 % Nonidet P-40)
Pfu-X Buffer (green cap)
10x conc.
Recommended 50 μl PCR assay:
5 μl | 10x Pfu-X Buffer | green cap |
200 μM | each dNTP | - |
0.4 μM | each Primer | - |
1 - 100 ng | template DNA | - |
0.5 μl (1.25 units) | Pfu-X Pol | red cap |
Fill up to 50 μl | PCR-grade water | - |
Please note that it is essential to add the polymerase as last component.
Recommended cycling conditions:
Three-step standard protocol
initial denaturation | 95 °C | 2 min | 1x |
denaturation | 95 °C | 20 sec | 25-30x |
annealing1) | 50 - 68 °C | 30 sec | 25-30x |
elongation2) | 68 °C | 1 min/kb | 25-30x |
final elongation | 68 °C | 1 min/kb | 1x |
Two-step protocol for amplification of longer fragments (>3 kb)
Please note that for performing two-step cycling a sufficiently high primer Tm is necessary. If Tm of primers is below 65 °C or two-step PCR does not yield a sufficient product quality the three-step cycling protocol is recommended.
initial denaturation | 95 °C | 2 min | 1x |
denaturation | 95 °C | 20 sec | 25-30x |
annealing/ elongation1,2) | 68 °C | 30 sec/kb | 25-30x |
final elongation | 68 °C | 30 sec/kb | 1x |
1) The annealing temperature depends on the melting temperature of the primers used.
2) 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.
Produtos relacionados:
- Ready-to-Use Mixes / direct gel loading
- Ready-to-Use Mixes
- Thermophilic Polymerases
- Deoxynucleotides (dNTPs)
- Supplements
- Primers and Oligonucleotides
- DNA Ladders