- Производитель:
- Roche
Enzyme Commission (EC) Number: | 2.7.7.7 ( BRENDA | IUBMB ) |
Кат. номер |
11644955001 |
11644947001 |
- Due to its proofreading activity, the thermostable Pwo DNA Polymerase has an extremely low error rate, 18-fold lower compared to Taq DNA Polymerase. It is therefore ideal for applications that require the highest possible fidelity in DNA synthesis. It can be applied for High fidelity PCR
- Cloning of PCR products
- Characterization of rare mutations
- PCR
Features and Benefits
- Excellent accuracy (18-fold more accurate than Taq DNA polymerase)
- High thermal stability
- Nearly as processive as Taq DNA polymerase
- Accepts modified nucleotides
Packaging
1 kit containing 4 components
Quality
Each lot of Pwo DNA polymerase is assayed for:
- Activity: The enzyme is tested on activated DNA.
- Function: The enzyme is tested in two PCRs, using DNA and human genomic DNA as templates.
- Proofreading ability: Proofreading activity is assayed according to the laq Iq fidelity assay [Frey, B. & Suppmann, B. (1995) Biochemica 2. 8-9].
- Absence of nucleases: The enzyme is tested on various substrates to ensure the absence of detectable endonucleases, exonucleases, and nicking activity according to the current Quality Control procedures.
Unit Definition
One unit Pwo DNA polymerase is defined as the amount of enzyme that catalyzes the incorporation of 10 nmol total deoxynucleoside triphosphates into acid precipitable DNA within 30 minutes at +70 °C under the conditions described below.
Unit Assay: Incubation buffer for assay on activated DNA
20 mM Tris-HCl, pH 8.8 (20 °C), 50 mM KCl, 2.5 mM MgCl2, 10 mM 2-mercaptoethanol, 0.2 mM of each dATP, dCTP, dGTP, dTTP.
Incubation procedure
12.5 mg activated calf thymus DNA and 0.1 mCi [-32P]dCTP are incubated with 0.01 to 0.1 U Pwo DNA Polymerase in 50 µl incubation buffer with a paraffin oil overlay at +70 °C for 30 minutes. The amount of incorporated dNTPs is determined by trichloroacetic acid precipitation followed by scintillation counting.
Volume Activity: 5 U/µl
Preparation Note
Pwo DNA polymerase was originally isolated from Pyrococcus woesei, a hyperthermophilic archaebacterium.
Modified nucleotides are substrates
Pwo DNA Polymerase accepts modified nucleotides like digoxigenin-dUTP, biotin-dUTP, or fluorescein-dUTP. Thus, it can add these nucleotides to DNA during PCR. These nonradioactively labeled products can be used as a hybridization probe in many applications.
Magnesium concentration
If you use the magnesium-containing reaction buffer supplied with the enzyme, the final MgCl2 concentration in the PCR will be 2.0mM. For other magnesium concentrations (e.g., for optimizing the reaction to accommodate a particular template), use the magnesium-free reaction buffer and add appropriate amounts of the magnesium stock.
usage | sufficient for ≤200 reactions (11644955001), sufficient for ≤40 reactions (11644947001) |
Quality Level | 100, |
feature | High Fidelity PCR, dNTPs included: no, hotstart: no |
packaging | pkg of 100 U (11644947001), pkg of 500 U (11644955001 [2 x 250 U]) |
Торговая марка | Roche |
application(s) | PCR: suitable |
shipped in | dry ice |
storage temp. | −20°C |
RIDADR | NONH for all modes of transport |
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