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Description
Pfu DNA Polymerase ⅡProduct Specification Synonyms DNA polymerase DNA polymerase BPfu polymerasePol I Expression System E. coli Molecular Weight 90 kDa (Reducing) Purity 95% by SDS PAGE Tag His Tag Physical Appearance Liquid Storage Buffer 20 mM Tris HCl, 0. 1 mM EDTA, 0. 1% Tween20, 0. 1% triton X100, 1 mM DTT, 100 mM KCl, 50%GlycerolpH 8. 2 @ 25C Stability & Storage Store at 25 ~ 15 for 2 years Reference [1] Lin, T. C. , et al. "Cloning and expression of T4 DNA
Product Specification
| Synonyms | DNA polymerase、 DNA polymerase B、Pfu polymerase、Pol I |
| Expression System | E.coli |
| Molecular Weight | 90 kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE |
| Tag | His Tag |
| Physical Appearance | Liquid |
| Storage Buffer | 20 mM Tris-HCl, 0.1 mM EDTA, 0.1% Tween20, 0.1% triton X100, 1 mM DTT, 100 mM KCl, 50%Glycerol、pH 8.2 @ 25°C |
| Stability & Storage | Store at -25 ~ -15℃ for 2 years |
| Reference | [1] Lin, T. C. , et al. "Cloning and expression of T4 DNA polymerase." Proceedings of the National Academy of Sciences 84.20(1987):7000-7004. |
Background
Pfu DNA Polymerase is a thermostable enzyme that replicates DNA at 75°C. It catalyzes the polymerization of nucleotides into duplex DNA in the 5´→3´ direction in the presence of magnesium. The enzyme has a molecular weight of approximately 90,000 daltons as estimated from the predicted amino acid sequence and exhibits 3´→5´ exonuclease (proofreading) activity. Pfu DNA Polymerase is recommended for use in PCR and primer extension reactions that require high fidelity.
Components
Storage Solution : 5 U/ul Pfu DNA Polymerase Ⅱ, 20 mM Tris-HCl, 0.1 mM EDTA, 0.1% Tween20, 0.1% triton X100, 1 mM DTT, 100 mM KCl, 50%Glycerol、pH 8.2 @ 25°C
10* Reaction Buffer: 200 mM Tris-HCl (pH 8.8) , 20 mM MgSO4, 100 mM KCl, 100 mM (NH4) 2SO4, 1% Triton X-100, 1 mg/ml nuclease-free BSA
Protocol
1. Set up a 50 μl PCR reaction system as follows:
Reagent |
Volume |
Final Concentration |
10X Pfu Buffer (with Mg2+) |
5µl |
1 X |
dNTP mix, 10mM each |
1µl |
200µM each |
upstream primer |
5–50pmol |
0.1–1.0µM |
downstream primer |
5–50pmol |
0.1–1.0µM |
DNA template |
variable |
<0.5µg/50µl |
Pfu DNA Polymerase (5U/µl) |
variable |
1.25U/50µl |
DEPC-treated Water |
Up to 50µl |
- |
2. Recommended thermal cycling conditions for Pfu DNA Polymerase-mediated PCR amplification as follows:
Step |
Temperature |
Time |
Number of Cycles |
Initial Denaturation |
95°C |
1–2 minutes |
1 cycle |
|
Denaturation Annealing* Extension |
95°C 42–65°C 72–74°C |
0.5–1 minute 30 seconds 2–4 minutes |
25–35 cycles
|
Final Extension |
72–74°C |
5 minutes |
1 cycle |
Soak |
4°C |
Indefinite |
1 cycle |
*The annealing temperature for a specific amplification reaction will depend upon the sequences of the two primers.
Guidelines
Note: It is critical to withhold Pfu DNA Polymerase until after the addition of dNTPs; otherwise, the proofreading activity of the polymerase may degrade the primers,resulting in nonspecific amplification and reduced product yield. Assemble on ice.
Unit Definition
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