11480022001

Tth DNA Polymerase

pkg of 500 U (2 x 250_U), sufficient for ≤200 reactions

• 동의어(들): DNA polymerase, polymerase
• 효소 위원회 번호: 2.7.7.7 (BRENDA, IUBMB)
• UNSPSC 코드: 41106314

속성

• 생물학적 소스: bacterial (Thermus thermophilus)
• Quality Level: 100
• 재조합: expressed in E. coli
• 양식: liquid
• 사용: sufficient for ≤200 reactions
• 특이 활성도: 5 U/μL
• 포장: pkg of 500 U (2 x 250_U)
• 제조업체/상표: Roche
• 농도: 0.5-5.0 units (per reaction for PCR (standard)); 2.5 units (per reaction for PCR (standard))
• 파라미터: 75 °C optimum reaction temp.
• 기술: PCR: suitable; RT-PCR: suitable; nucleic acid labeling: suitable
• 색상: colorless
• 최적 pH: ~9.0 (25 °C)
• solubility: water: miscible
• 적합성: suitable for molecular biology
• UniProt 수납 번호: P30313
• 응용 분야: life science and biopharma
• 외래 활성: Endonuclease, none detected (up to 20 enzyme units using Lambda-DNA/ 16h/37°C); Nicking activity, none detected ( up to 20 enzyme units using pBR322-DNA / 16h/37°C); RNases, none detected (up to 20 enzyme units using MS II-RNA; 4h/37°C)
• 저장 온도: −20°C

설명

일반 설명

Tth DNA Polymerase is isolated from the thermophilic eubacterium Thermus thermophilus HB8, and is purified to be free of nonspecific DNases and RNases. The enzyme is a highly processive 5′-3′ DNA polymerase, and lacks 3′-5′ exonuclease activity. The enzyme exhibits its highest activity at a pH of approximately 9 (adjusted at +25°C), and temperatures around +75°C. It is resistant to prolonged incubations at high temperatures (+95°C).

애플리케이션

Thermus thermophilus (Tth) DNA Polymerase might be used:

• to amplify DNA fragments by polymerase chain reaction (PCR) due to its resistance towards prolonged incubations at high temperatures (95 °C)
• to label DNA fragments with either radiolabeled nucleotides, digoxigenin, or biotin, since this enzyme accepts modified deoxyribonucleotides as substrates
• to efficiently transcribe RNA targets into cDNA due to its intrinsic Mn-dependent reverse transcriptase (RT) activity
• for real time PCR

생화학적/생리학적 작용

Thermus thermophilus (Tth) DNA Polymerase is a thermostable DNA polymerase with intrinsic reverse transcriptase (RT) activity. The enzyme is a highly processive 5′-3′ DNA polymerase and lacks 3′-5′ exonuclease activity (proof reading). It was found to possess a very efficient intrinsic reverse transcriptase (RT) activity in the presence of manganese ions – much higher than that demonstrated for Escherichia coli DNA polymerase I and Taq DNA Polymerase. The RT activity is not associated with RNase H activity. The elevated temperatures of Tth DNA Polymerase (optimum +55°C to +70°C, maximum +95°C) activity overcomes the problems posed by RNA secondary structure. Resulting cDNA can be amplified by PCR using the same enzyme in the presence of Mg²⁺-ions. The ability of Tth DNA Polymerase to perform both reverse transcription and DNA amplification at elevated temperatures allows this enzyme to be used for quantitative RT-PCR, cloning, and gene expression analysis of cellular and viral RNA. Tth DNA Polymerase is used for RT-PCR amplification of RNA up to 1kb.

특징 및 장점

Tth DNA Polymerase:

• Ensures optimized polymerase chain reaction (PCR) product size for at least up to 1,000 bp in a RT-PCR reaction
• Accepts modified desoxyribonucleoside triphosphates as substrates
• Has no association with RNase H activity
• Has high thermostability to overcome the problem, typically associated with the high degree of secondary structure present in RNA

포장

1 kit containing 3 components

품질

Each lot of Tth DNA Polymerase is assayed for activity on activated DNA. A function test is performed using ?DNA, as well as human genomic DNA. Performance in RT/PCR is assayed using total mouse RNA and primers specific for ?-actin. Each lot of Tth DNA Polymerase is tested for the absence of contaminating activities like exo- and endonucleases and nicking activities according to the current Quality Control procedures. A unique self-priming assay ensures the absence of contaminating DNA according to the current Quality Control procedures.

단위 정의

One unit of Tth DNA Polymerase is defined as the amount of enzyme which catalyses the incorporation of 10 nmol total dNTPs into acid precipitable DNA within 30 minutes at +70 °C under the assay conditions stated in "unit assay".

Unit Assay: Incubation buffer for assay on activated DNA
67 mM Tris-HCI, pH 8.8 (+25 °C), 16.6 mM (NH₄)₂SO₄, 6.7 mM MgCl₂, 10 mM 2-mercaptoethanol, 0.2 mM dATP, dCTP, dGTP, dTTP each.

Incubation procedure
12.5 μg activated herring sperm DNA and 0.1 μCi [α³²P] dCTP are incubated with 0.01 to 0.1 units Tth DNA Polymerase in 50 μl Incubation buffer with a paraffin oil overlay at +70 °C for 30 min.
The amount of incorporated dNTPs is determined by trichloroacetic acid precipitation followed by scintillation counting.

Volume Activity: 0.5 to 5 U per reaction of PCR (optimal)
2.5 U per reaction of PCR (standard)
Determined in the assay on activated DNA described under "unit assay".

기타 정보

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

법적 정보

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 such patent claims for using only this amount of product for the purchaser′s own internal research. No right under any other patent 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.

키트 구성품 전용

• PCR Buffer, including MgCl₂ 10x concentrated
• RT-PCR Buffer, coupled reaction in one tube 5x concentrated
• Mn(OAc)₂-Solution 25 mM

안전성 정보

• Storage Class Code: 12 - Non Combustible Liquids
• WGK: WGK 1
• Flash Point (°F): does not flash
• Flash Point (°C): does not flash