- Description
- Specifications
Catalog #: ENZ-265
Description:
Pfu DNA Polymerase is a thermo-stable enzyme having a Mw of about 90kDa.
Pfu DNA Polymerase is derived from E. coli that and cloned from Pyrococcus furiosus strain Vc1 DSM3638. Pfu DNA Polymerase replicates DNA at 75 °C, catalyzing the polymerization of nucleotides into duplex DNA in the 5 ´ to 3 ´ direction in the existence of magnesium. Pfu DNA Polymerase possesses 3 ´ to 5 ´ exonuclease (proofreading) activity. Base misinsertions that take place during polymerization are swiftly removed by the proofreading activity of the polymerase. Therefore, Pfu DNA Polymerase is suggested for use in PCR and primer extension reactions that require high-fidelity synthesis. Pfu DNA Polymerase-generated PCR fragments are blunt-ended.
Synonyms:
DNA polymerase, EC 2.7.7.7, Pfu polymerase, Pfu-DNA Polymerase.
Source:
Escherichia Coli.
Applications:
1. Ideal for high-fidelity amplification.
2. 3'-5' exonuclease activity provides a low error rate.
3. One of the most thermostable DNA polymerases known.
4. Lack of extendase activity means no unwanted 3' overhangs.
5. Optimal for blunt-end PCR cloning.
6. Optimum temperature near 75 °C.
7. 95% active after 1-hour incubation at 98 °C.
Formulation:
50mM Tris-HCl, pH 8.2, 1mM DTT, 0.1mM EDTA, 0.05% CHAPS and 50% glycerol.
Stability:
Pfu DNA Polymerase although stable at 10 °C for 5 days, should be stored below -18 °C.
Please prevent freeze-thaw cycles.
Physical Appearance:
Sterile liquid formulation.
10X Buffer with MgSO4:
200mM Tris-HCl (pH 8.8 at 25 °C), 100mM (NH4)2SO4, 100mM KC1, 1% Triton X-100, 1mg/ml BSA, 20mM MgSO4.
PCR Protocol:
Add the following components to a amplify 1kb DNA template:
5 µl 10x buffer with MgSO4.
4 µl 2.5mM dNTPs.
1 µl Primers mix (10 µM each).
0.2 µl Pfu-DNA Polymerase.
and 38 µl Water.
Amplify using the following cycling parameters:
Heat Soak: 1 cycle at 94 °C/4 min.
Denaturation: 30 cycles at 94 °C/30 sec.
Annealing: 30 cycles at 58 °C /30 sec.
Extension: 30 cycles at 72 °C /90 sec.
Final: 1 cycle at 72 °C /5 min.
Unit Definition:
1U of enzyme catalyzes the incorporation of 10nmol of dNTP into acid-insoluble product in 30 minutes at 75 °C.
Usage:
Denovo Biotechnology's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.
