Certificate of Analysis and Data Sheet

Source:
E. coli

Catalog No.
ENZ-291

Description :

Recombinant Pfu DNA Polymerase is purified from an E.coli strain carrying a plasmid with the cloned gene encoding the hyperthermophilic archae Pyrococcus furiosus DNA Ploymerase 92KDa. Pfu DNA Polymerase is referred from here on as Pfu. Pfu has been shown to exhibit superior thermostability and proofreading properties compared to other thermostable polymerase. Unlike Taq DNA Polymerase, highly thermostable Pfu possesses 3' to 5' exonuclease proofreading activity that enables the polymerase to correct nucleotide-misincorporation errors. This means that Pfu generated PCR fragments will have fewer errors than Taq-generated PCR inserts. The error rate for Pfu is reported to be 7 to 10 fold lower than that of nonproofreading Taq DNA polymerase, and 2 to 30 fold lower than other proofreading enzymes. Using Pfu in your PCR reactions results in blunt-ended PCR products, which are ideal for cloning into blunt-ended vectors, such as the PCR-Script?vectors. Pfu is superior for techniques that require high-fidelity DNA synthesis. The enzyme catalyzes the incorporation of nucleotides into duplex DNA in the 5?>3?direction in the presence of Mg2+ at 70蚓- 80蚓. Pfu DNA Polymerase exhibits 3?>5?exonuclease (proofreading) activity, but has no detectable 5?>3?exonuclease activity.

Concentration:

2.5U/痞.

Unit Definition:

One unit of enzyme catalyzes the incorporation of 10 nanomoles of deoxyribonucleotides (dNTP) into a polynucleotide fraction (adsorbed on DE-81) in 30min at 72蚓.

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蚓. 7. 95% active after 1-hour incubation at 98蚓.

Storage Buffer:

20mM Tris-HC1 (pH 8.2), 1mM DTT, 0.1mM EDTA, 100mM KC1, 0.1% Nonidet P40, 0.1% Tween 20 and 50% glycerol.

Stability:

Pfu although stable at 4⁰C for 2 weeks, should be stored desiccated below -18⁰C. Please prevent freeze-thaw cycles.

10X PCR Buffer with MgSO4:

200mM Tris-HC1 ( pH 8.8 at 25蚓), 100mM (NH4)2SO4, 100mM KC1, 1% Triton X-100, 1mg/ml BSA, 20mM MgSO4.

Usage:

This material is offered by ProSpec-TechnoGene for research, laboratory or further evaluation purposes.

Latest Publications:

1. Addendum: Site-directed mutagenesis using Pfu DNA polymerase and T4 DNA ligase.
Biotechniques 2005 Sep;39(3):328
2. Site-directed mutagenesis using Pfu DNA polymerase and T4 DNA ligase.
Biotechniques 2005 Jun;38(6):864, 866, 868
3. A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase.
Biotechnol Appl Biochem 2005 Dec;42(Pt 3):223-6
4. Improved antibiotic-resistance cassettes through restriction site elimination using Pfu DNA polymerase PCR.
Biotechniques 1998 Nov;25(5):772-4, 776
5. Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence.
Genome Res 1997 Aug;7(8):843-52
6. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.
Nucleic Acids Res 1996 Sep 15;24(18):3546-51