Pfu DNA Polymerase is a thermostable DNA polymerase isolated from an E. coli strain that carries the pol gene from Pyrococcus furiosus. As one of the most thermostable DNA polymerases known to have 3’ to 5’ proofreading activity, the enzyme catalyzes DNA systhesis at optimal temperature near 75°C with very low error rate; it is about eight times more accurate than Taq DNA polymerase. Pfu DNA polymerase generates blunt-ended PCR fragments and so may be useful for projects that involve blunt-end PCR cloning.
Size: 250 units.
Source:
Escherichia coli.
Physical Appearance:
Sterile liquid formulation.
Formulation:
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 DNA Polymerase although stable at 10°C for 5 days, should be stored desiccated below -18°C.
Please prevent freeze-thaw cycles.
Unit Definition:
1U of enzyme catalyzes the incorporation of 10nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 minutes at 72°C.
10X PCR Buffer with MgSO4:
200mM Tris-HCl (pH 8.8 at 25°C), 100mM (NH4)2SO4, 100mM KC1, 1% Triton X-100, 1mg/ml BSA, 20mM MgSO4.
PCR Protocol:
1. Add the following component to a PCR tube at ambient temperature or on ice:
Template DNA (1-200ng): 1µl
10x PCR buffer: 5µl
10mM dNTPs: 1µl
Primers mix (10M each): 2µl
Pfu-DNA Polymerase: 1µl
Water: 40µl
Total Volume 50µl
For greater than 1kb target 1U is sufficient.
When amplifying targets greater than 3kb, 2.5U may be required.
2. Amplify using the following cycling parameters:
1x cycle: 95°C for 2 minutes
30x cycles: 95°C, 30 sec; 55°C, 30 sec; 72°C for 1 minute/1kb (2 or 3 minutes per kb for PCR product >3 kb).
1x cycle: 72°C for 7 minutes.
3. Remove 10 µl from reaction and analyze by agarose gel electrophoresis.
QC
PCR amplification of 300 bp to 1 kb fragments using Pfu DNA Polymerase
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°C.
7. 95% active after 1-hour incubation at 98°C.
This product is not available in some countries (for example, USA).
