Hot start Pfu DNA polymerase is formulated with chemical modification which effectively neutralize 5´→ 3´ DNA polymerase and 3´→ 5´ exonuclease (proofreading) activities at room temperature, but regain the full enzyme activity upon the initial denaturation step. Hot start Pfu DNA polymerase retains the high fidelity, sensitivity and processivity of Pfu DNA polymerase, while provides reduced background by facilitating room temperature PCR assembly and preventing priming until stringent primer annealing temperatures are reached. Pfu DNA polymerase has an error rate six-fold lower than Taq DNA polymerase, and significantly lower than the error rates of most other proofreading enzymes or DNA polymerase mixtures (1). This product is supplied with the unique Intact Genomics 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM, and 5x Magic Enhancer that enables efficient amplification of GC rich templates up to 84%.
The physical purity of this enzyme is ≥98% as assessed by SDS-PAGE with Coomassie® blue staining (see figure below).
E. coli strain expressing a Pfu DNA Polymerase gene from Pyrococcus furiosus.
Hot start Pfu Polymerase data:
A typical gel data with Hot Start Pfu Polymerase for PCR assay is shown below in Fig. 2.
- Routine PCR
- Primer extension
- Colony PCR
- Efficient for amplifying high GC content template DNA with Magic Enhancer
- Hot Start Pfu DNA Polymerase
- 10x PCR Buffer with Mg2+
- 5x Magic Enhancer
- 10 mM dNTP Mix (Cat. # 3316d, 3318d only)
50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 ºC.
10X PCR Buffer with Mg2+
100 mM Tris-HCl pH 9.0, 15 mM MgCl2, 100 mM KCl , 80 mM (NH4)2SO4, 0.5% Igepal CA 630
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTP into acid-insoluble form in 30 minutes at 72 ºC.
- Thaw 10x PCR Buffer, dNTP mix, primer solutions, 5X Magic Enhancer (if required) and mix thoroughly before use.
- Prepare a reaction mix according to the following table: The reaction mix typically contains all the components needed for PCR except the template DNA.
- Mix the reaction mixture thoroughly.
- Add template DNA to the individual PCR tubes containing the reaction mixture.
- Program the thermal cycler according to the manufacturer’s instructions. A typical PCR cycling program is outlined in the following table.
Note: PCR program must start with an initial heat-activation step at 95 °C for 15 min.
- Place the PCR tubes in the thermal cycler and start the cycling program.
- Frey, B. and Suppman, B. (1995). BioChemica. 2, 34-35.