PWOPOL-RO

Pwo DNA Polymerase

• Synonym(s): polymerase, dna, pwo
• Enzyme Commission number: 2.7.7.7 (BRENDA, IUBMB)
• UNSPSC Code: 41106300

Properties

• biological source: microbial (Pyrococcus woesei)
• Quality Level: 100
• form: liquid
• usage: sufficient for ≤200 reactions (11644955001); sufficient for 200 reactions; sufficient for ≤40 reactions (11644947001); sufficient for 40 reactions
• specific activity: ≥5000 U/mL
• mol wt: 90 kDa
• feature: High Fidelity PCR; dNTPs included: no; hotstart: no
• packaging: pkg of 100 U (11644947001); pkg of 500 U (11644955001 [2 x 250 U])
• manufacturer/tradename: Roche
• storage condition: (Containers which are opened must be carefully resealed and; kept upright to prevent leakage)
• concentration: 2.5 units/reaction
• technique(s): PCR: suitable
• color: colorless
• input: purified DNA
• solubility: water: soluble
• suitability: suitable for enzyme test
• application(s): genomic analysis; life science and biopharma
• foreign activity: Endonucleases with lambda-DNA 30 units, none detected; Nicking act using pBR322-DNA ≤30 units, none detected
• storage temp.: −20°C

Description

General description

Pwo DNA Polymerase is a highly processive thermostable 5′-3′ DNA polymerase and possesses a 3′–5′ exonuclease activity, also known as proofreading activity. The enzyme has no detectable 5′–3′ exonuclease activity. Pwo DNA Polymerase exhibits increased thermal stability with a half-life of more than two hours at +100 °C, compared to Taq DNA Polymerase′s half-life of less than 5 minutes at this temperature. Pwo DNA Polymerase-generated PCR products are blunt-ended and can therefore be used directly for blunt-end ligation without any pretreatment of the ends.
The use of Pwo DNA Polymerase during PCR significantly reduces the occurrence of random amplification errors.

Application

• 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 λDNA 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 [α-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.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Legal Information

Use of this product is covered by US patent claims and corresponding patent claims outside the US. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser′s own internal research. No right under any other patent claim, no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser′s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patent claims require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.</ br>NOTICE TO PURCHASER: LIMITED LICENSE<br />Use of this product is covered by US Patent No. 6,127,155 and corresponding patent claims outside the US.  The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser′s own internal research.  No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser′s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel.  This product is for research use only. Human and veterinary diagnostic uses under Roche patent claims require a separate license from Roche.  All uses other than internal research and human and veterinary diagnostic uses under Roche patent claims require a separate license from Thermo Fisher Scientific. Further information on purchasing licenses from Roche may be obtained by contacting the Licensing Department of Roche Molecular Systems, Inc., 4300 Hacienda Drive, Pleasanton, California 94588, USA or Roche Diagnostics GmbH, Sandhofer Strasse 116, 68305 Mannheim, Germany.  Further information on purchasing licenses from Thermo Fisher Scientific may be obtained by contacting the Licensing Department of  Thermo Fisher Scientific, 5791 Van Allen Way, Carlsbad, California 92008, USA.

Kit Components Only

• Enzyme is supplied in storage and dilution buffer
• PCR buffer, with 20 mM MgSO₄ 10x concentrated
• PCR buffer, without MgSO₄ 10x concentrated
• MgSO₄ stock solution

Safety Information

• Hazard Statements: H412
• Precautionary Statements: P273 - P501
• Hazard Classifications: Aquatic Chronic 3
• Storage Class Code: 12 - Non Combustible Liquids
• WGK: WGK 2
• Flash Point(F): does not flash
• Flash Point(C): does not flash