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Pfu DNA Polymerase (not available in the USA)

Pfu DNA Polymerase (native)		Pfu DNA Polymerase (recombinant)
#EP0571 Supplied with: 10X Pfu Buffer with MgSO4 10X Pfu Buffer 25 mM MgSO4	100 u  (2.5 u/µl) 0.6 ml  0.6 ml 0.6 ml	#EP0501 Supplied with: 10X Pfu Buffer with MgSO4 10X Pfu Buffer 25 mM MgSO4	100 u  (2.5 u/µl) 0.6 ml  0.6 ml 0.6 ml
#EP0572 Supplied with: 10X Pfu Buffer with MgSO4 10X Pfu Buffer 25 mM MgSO4	500 u (2.5 u/µl) 2x1.25 ml  2x1.25 ml 2x1.25 ml	#EP0502 Supplied with: 10X Pfu Buffer with MgSO4 10X Pfu Buffer 25 mM MgSO4	500 u (2.5 u/µl) 2x1.25 ml  2x1.25 ml 2x1.25 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis & MSDS:
#EP0571, #EP0572; MSDS (English) MSDS (English-USA) MSDS (German)		#EP0501, #EP0502; MSDS (English) MSDS (English-USA) MSDS (German)
Flyer (448 KB)
see Protocol for PCR with Pfu DNA Polymerase

 

Features

• Eight times more accurate than Taq DNA polymerase.
• Highly thermostable - remains 95% active after 2 hours incubation at 95°C.
• Generates blunt-end PCR products.
• Incorporates modified nucleotides (e.g., biotin-, digoxigenin-, fluorescently-labeled nucleotides).
• Increased PCR yields with native Pfu DNA Polymerase.

Pfu DNA Polymerase. PCR Enzyme Mixes for Long and Accurate PCR, flyer in pdf, 448 KB

Accuracy of thermostable DNA polymerases.

Description
The Pfu DNA Polymerase is a highly thermostable DNA polymerase from the hyperthermophilic archaeum Pyrococcus furiosus. The enzyme catalyzes the template-dependent polymerization of nucleotides into duplex DNA in the 5'=>3' direction. The Pfu DNA Polymerase also exhibits 3'=>5' exonuclease (proofreading) activity, that enables the polymerase to correct nucleotide incorporation errors. It has no 5'=>3' exonuclease activity.

Source
Pfu DNA Polymerase (native) - Pyrococcus furiosus cells.
Pfu DNA Polymerase (recombinant) - E.coli cells with a cloned pol gene from Pyrococcus furiosus.

Molecular Weight
90 kDa monomer.

Applications

• High fidelity PCR, see protocol.
• Blunt-end PCR cloning (1).
• Site-directed mutagenesis.
• Native Pfu DNA Polymerase is recommended for greater PCR yields.

Quality Control
The absence of endodeoxyribonucleases and exodeoxyribonucleases confirmed by appropriate quality tests. Functionally tested in PCR.

Concentration
2.5 u/µl

Definition of Activity Unit
One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 min at 72°C.
Enzyme activity is assayed in the following mixture: 20 mM Tris-HCl (pH 8.8 at 25°C), 2 mM MgSO₄, 10 mM (NH₄)₂SO₄, 10 mM KCl, 0.1% (v/v) Triton X-100, 0.1 mg/ml BSA, 0.75 mM activated calf thymus DNA, 0.2 mM of each dNTP, 0.4 MBq/ml [³H]-dTTP.

Storage Buffer
The enzyme is supplied in: 20 mM Tris-HCl (pH 8.2), 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, 0.1% (v/v) Nonidet P40, 0.1% (v/v) Tween 20 and 50% (v/v) glycerol.

10X Pfu Buffer with 20 mM MgSO₄
200 mM Tris-HCl (pH 8.8 at 25°C), 100 mM (NH₄)₂SO₄, 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA and 20 mM MgSO₄.

10X Pfu Buffer
200 mM Tris-HCl (pH 8.8 at 25°C), 100 mM (NH₄)₂SO₄, 100 mM KCl, 1% (v/v) Triton X-100, 1 mg/ml BSA.

Inhibition and Inactivation
Inactivated by phenol/chloroform extraction.

Note

• The error rate of Pfu DNA Polymerase in PCR is 2.6x10^-6 errors per nt per cycle, as determined by a modified method described in (2).
• Accordingly, the accuracy of PCR is 3.8x10⁵. Accuracy is an inverse of the error rate and shows an average number of correct nucleotides incorporated before an error occurs.
• The Pfu DNA Polymerase has no detectable reverse transcriptase activity.
• dUTP, dITP and primers containing these nucleotides should not be used in PCR with Pfu DNA Polymerase because the binding of this enzyme to DNA templates with uracil and hypoxanthine stalls DNA synthesis (3, 4).

Amplification of 950 bp single copy gene from human genomic DNA using Pfu DNA Polymerase (native). A 950 bp single copy gene from human genomic DNA was amplified using 0.5-2.5 u of the enzyme in 50 µl of reaction mixture.
M - GeneRuler™ 100 bp Plus DNA Ladder
1 -5 - PCR products generated with different amounts of Pfu DNA Polymerase

 

Related Products

dNTP Mixes dNTP Set Modified Nucleotides (molecular biology grade): Aminoallyl-dUTP  Biotin-11-dUTP Fluorescein-12-dUTP dm6ATP dm4CTP dm5CTP CloneJET™ PCR Cloning Kit FastRuler™ DNA Ladders, ready-to-use O'RangeRuler™ DNA Ladders, ready-to-use GeneRuler™ DNA Ladders Genomic DNA Purification Kit DNA Gel Extraction Kit Agarase Water, nuclease-free

References

1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.

2. Lundberg, K.S., et al., High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus, Gene, 108, 1-6, 1991.
3. Shuttleworth, G., et al., Recognition of the pro-mutagenic base uracil by family B DNA polymerases from Archaea, J. Mol. Biol., 337, 621-634, 2004.
4. Gruz, P., et al., Processing of DNA lesions by archaeal DNA polymerases from Sulpholobus solfataricus, Nucleic Acids Res., 31, 4024-4030, 2003.

Updated kovo 18, 2008 09:54