• Pfu DNA Polymerase
  • Pfu DNA Polymerase

Pfu DNA Polymerase

Pfu DNA Polymerase is one of the most commonly used high-fidelity DNA polymerase, a highly thermally stable DNA polymerase derived from the thermophilic bacterium Pyrococcus furiosus. The molecular weight of Pfu enzyme is 90kDa. Pfu enzymes can catalyze the polymerization of DNA template dependent deoxynucleotides in the 5 'to 3' direction
$34.00
  • Pfu DNA Polymerase

SPECIFICATION


                      Pfu DNA Polymerase

Pfu DNA Polymerase is one of the most commonly used high-fidelity DNA polymerase, a highly thermally stable DNA polymerase derived from the thermophilic bacterium Pyrococcus furiosus. The molecular weight of Pfu enzyme is 90kDa. Pfu enzymes can catalyze the polymerization of DNA template dependent deoxynucleotides in the 5 'to 3' direction. Unlike Taq enzyme, Pfu enzyme has exonuclease activity ranging from 3 'to 5', which greatly reduces the probability of errors during PCR amplification, with an error rate of 2.6 × 10-6 per nt per cycle. The error rate of Pfu is not only much lower than that of Taq enzyme, but also lower than some other high-fidelity DNA polymerase enzymes such as Vent, DeepVent, Pwo, Tli, etc. Therefore, Pfu enzyme is often chosen as the preferred cost-effective high-fidelity DNA polymerase. In addition, Pfuase has 5 'to 3' exonuclease activity, but Pfuase does not reverse transcriptase activity.
High fidelity PCR, point mutation, double ended PCR cloning, etc.
The DNA fragments amplified by Pfu enzyme are double ended and can be used for double ended cloning, but cannot be used for conventional T-vector cloning.
DUTP and dITP, or primers containing dUTP and dITP, cannot be used for Pfu enzyme mediated PCR amplification reactions.
One unit of the enzyme catalysts of incorporation of 10nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) for 30 minutes at 72 ℃
Activity detection conditions:
20mM Tris HCl (pH8.8 at 25 ℃), 2mM MgSO4, 10mM (NH4) 2SO4, 10mM KCl, 0.1% (v/v) Triton X-100, 0.1mg/ml BSA, 0.75mM activated calf thymus DNA, 0.2mM of each dNTP, 0.4 MBq/ml [3H] - dTTP
This Pfu DNA Polymerase is a recombinant Pfu DNA Polymerase obtained through expression and purification in Escherichia coli, and has the same properties as the purified natural Pfu DNA Polymerase in various aspects.
Component 200U 1000U
Pfu DNA Polymerase (5U/ μ L) 40 μ L 200 μ L
ten × Pfu Buffer (with Mg2+) 1mL 5 × 1mL
Storage: -20 ℃

Setting up the PCR reaction system:
Dissolve and mix the various solutions required for PCR reaction. Place Pfu DNA Polymerase on an ice bath or in an ice box.
Refer to the table below to set up PCR reactions on an ice bath (if there are multiple similar PCR reactions, a large volume mixture containing water, buffer, dNTP, and Pfu enzyme can be prepared first, and then packaged into each PCR reaction tube. Depending on the situation, sometimes primers can be included in the mixture):
Final concentration and dosage of ingredients
Double steamed water or Milli Q water - (36.75 x) μ L
ten × Pfu Buffer (with Mg2+) 1 × five μ L
DNTP (2.5mM each) 0.2mM each 4 μ L
Template DNA 10pg-1 μ G * x μ L
Primer mixture (10 μ M each) 0.8 μ M 4 μ L
Pfu DNA Polymerase (5U/ μ L) 1.25U/50 μ L 0.25 μ L
Total volume -50 μ L
Note *: For different types of templates at 50 μ The recommended dosage in L reaction volume is as follows: mammalian genome DNA: 0.1-1 μ G; Escherichia coli genome DNA: 10-100ng; Plasmid DNA: 0.1-10ng. Excessive template DNA can easily lead to non-specific PCR products.
Gently blow and mix with a pipette or lightly Vortex, centrifuge at room temperature for a few seconds, and allow the liquid volume to converge at the bottom of the tube.
If the PCR instrument used has a hot cover, omit this step. If the PCR instrument does not have a hot cap, drop a drop of mineral oil (product number YT399) into the tube.
Place each set PCR reaction tube on the PCR instrument and start the PCR reaction.
The setting of PCR reaction parameters can refer to the following examples:
STEP1 (initial denaturation): 94 ℃ for 3 minutes
STEP2 (denaturation): 94 ℃ for 30 seconds
STEP3 (annealing): 55 ℃ for 30 seconds
STEP4 (extension): 72 ℃ for 2 minutes
STEP5 (cycles): Go To STEP2 for 30 cycles
STEP6 (final extension): 72 ℃ for 10 minutes
STEP7 (temporary storage): 4 ℃ for ever
The setting of PCR reaction needs to be based on different conditions such as template, primer, length of PCR product, and GC content, including temperature, time, and number of cycles.
The time setting for STEP4 (extension) needs to be based on the length of the PCR product, usually with an extension time of 2 minutes per kb of product. For example, if the length of the PCR product is 1kb, the extension time can be set to 2 minutes. If the length of the PCR product is 2kb, the extension time can be set to 4 minutes, and so on.
For the initial PCR, to ensure that the expected PCR products can be amplified as much as possible, the number of cycles can be set to 35. For those that require semi quantitative or quantitative analysis
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