Super Pfu DNA Polymerase (cloned)

Product Description:

Super Pfu DNA polymerase is isolated from the hyperthermophilic marine archaebacterium, Pyrococcus furiosus. The multifunctional thermostable enzyme possesses both 5’- to 3’- DNA polymerase and 3’- to 5’- exonuclease activity, which results in a 12-fold increase in fidelity of DNA synthesis over Pfu DNA polymerase. Super Pfu DNA Polymerase has a temp. optimum between 72°C and 78°C remains > 95% active following a one hour incubation at 95°C.

 

Reaction Mixture Set Up--
  • Gently vortex and briefly centrifuge all solutions after thawing.
  • Add components, in the following order, into a thin-walled PCR tube. Keep all components on ice. The following control PCR reactions should be run in parallel to ensure that the Super Pfu DNA polymerase is working properly.
Reagent Final Conc. Quantity Reagent Positive Control Negative Control
Water (PCR—Grade) --- variable Water (PCR—Grade) 32.8μl 33.8μl
10X Pfu reaction buffer 1X 5μl 10X Pfu reaction buffer 5μl 5μl
2.5mM dNTP mixture 200μM of each 4μl 2.5mM dNTP mixture 4μl 4μl
Primer I, forward 0.1-1μM variable Primer I (10μM), forward 1μl 1μl
Primer II, reverse 0.1-1μM variable Primer II (10μM), reverse 1μl 1μl
Pfu DNA polymerase 1-1.5u/50μl Variable Pfu DNA polymerase 0.2μl 0.2μl
Template DNA See note 1 Variable Control DNA Template --- ---
Total Volume --- 50μl Total Volume 50μl 50μl
  • Gently vortex the sample and briefly centrifuge to collect all drops from walls of the tube.
  • Overlay the sample with one-half of the total reaction volume of mineral oil or add an appropriate amount of wax. This step may be omitted if the thermo cycler is equipped with a heated lid.
  • . Place samples in a thermo cycler and start PCR.

Note for the Components of the Reaction Mixture:

  • Template DNA: Usually the amount of template DNA is in the range of 0.01- 1ng plasmid or phage DNA & 0.1-1μg for genomic DNA, for a total reaction mixture of 50μl.
  • Primers: The PCR primers are usually 15-30 nucleotides in length, longer primers provide higher specificity. The GC content of primer should be 40-60%. The primer should not be self-complementary or complementary to any other primer in the reaction mixture, and the melting temperature of flanking primers should not differ by more than 5°C. If the primer is shorter than 25 nucleotides, the approx. melting temperature, Tm is calculated using the formula as: Tm=4(G+C) + 2(A+T).
  • dNTPs: The final concentration of each dNTP in the reaction mixture is usually 200μM.
  • Super Pfu DNA polymerase: Usually 1-15u of Super Pfu DNA polymerase are used in the 50μl of reaction mix. Higher Super Pfu DNA polymerase concentrations may cause synthesis of nonspecific products. However, if inhibitors are present in the reaction mix (e.g., if the template DNA used is not highly purified), higher amounts of Pfu DNA polymerase (2-3u) may be necessary to obtain a better yield of amplification products.
  • Usually the extending step is performed at 70-75°C. Super Pfu DNA Polymerase exhibits lower extension rate than Taq DNA Polymerase, so 2min extension time is recommended for every 1 kb to be amplified.
  • Cycling conditions: Usually denaturation for 0.5-2min at 94-95°C is sufficient; the optimal annealing temperature is 5°C lower than the melting temperature of primer-template DNA duplex; Usually the extending step is performed at 70-75°C. Recommended extending time is l min for the synthesis of PCR fragments up to 2kb. When larger DNA fragments are amplified, the extending time is usually increased by 1 min for each 1kb.
  • Number of cycles: The number of PCR cycles depends on the amount of template DNA in the reaction mix and on the expected yield of the PCR product. For less than 10 copies of template DNA, 40 cycles should be performed. If the initial quantity of template DNA is higher, 25-35 cycles are usually sufficient.
  • Final extending step: After the last cycle, the samples are usually incubated at 72°C for 5-15min.