Pfu DNA Polymerase Ⅱ

Catalog Number: UA070072 Brand: UA BIOSCIENCE

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$164 USD

Size: 250U 1000U

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Product Details

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. [2] Yan, Wang , et al. "A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro." Nucleic Acids Research 3(2004):1197.

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

One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74°C

Picture

Bioactivity

In the experiment, the 773bp target fragment was amplified by PCR using PUC57 as the template. At the same time, competing enzymes were added to investigate the enzyme activity of Pfu DNA PolymeraseⅡ. As shown in the figure, this product has the following effects.
Lane 1 Negative Control (negative control with no added enzyme only);
Lane 2 UA-Pfu DNA Polymerase Ⅱ 0.5U;
Lane 3 UA-Pfu DNA Polymerase Ⅱ 1.25U;
Lane 4 UA-Pfu DNA Polymerase Ⅱ 2.5U;
Lane 5 UA-Pfu DNA Polymerase Ⅱ 5U;
Lane 6 Competing product A 5U;
Lane 7 Competing product B 5U;

SDS-PAGE

2μg (R: reducing condition, N: non-reducing condition).

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Pfu DNA Polymerase Ⅱ

Pfu DNA Polymerase Ⅱ