Hieff Canace^TM Uracil+ High-Fidelity DNA polymerase, is a new generation of high-fidelity DNA Polymerase based on Pfu DNA Polymerase. The Hieff Canace^TM Uracil+ high-fidelity DNA Polymerase enables rapid and accurate PCR reactions for complex templates. Its fidelity is improved significantly, and it completely avoids the amplification failure caused by using dUTP-containing primers/templates/dNTPs. The product is equipped with an optimized enzyme buffer and the addition of PCR enhancing components, making the enzyme highly efficient and adaptable to a wide range of templates, suitable for amplification of complex templates.

Product Components   

Shipping and Storage

The components are shipped with ice packs and can be stored at -20°C for 1 year.

Product Application

High-fidelity library amplification using dUTP-containing primers/templates/dNTPs.

Cautions

1. For your safety and health, please wear lab coats and disposable gloves for operation.

2. For research use only!

Instructions

Hieff Canace^TM Uracil+ High-Fidelity DNA polymerase is used in a slightly different way than conventional high-fidelity enzymes. Please read the instructions carefully before use.

1. Recommended reaction system

All operations should be carried out on ice. 2×Canace^TM Uracil+ PCR buffer should be well mixed after thawing. Before the system configuration, please preheat the PCR instrument. After the addition of the components, mix the sample well by a pipette and spin it down to the bottom of the tube, then place it in the preheated PCR instrument for amplification. After use, return all components to -20 °C for storage.

Table 1. PCR amplification reaction

[Notes]:  

1)Reagents: Sufficiently thaw each component before use;

2)Templates: The use of purified high-quality DNA templates can significantly improve the efficiency and success rate of amplification;

3)dNTPs: The recommended final dNTPs concentration is 200 μM. The dNTPs provided in the kit do not contain dUTP. If special circumstances require the preparation of dUTP-containing templates, additional dUTP can be added to a final concentration of 400 μM.

4)Polymerase concentration: 1 U/50 μL is recommended. It can be optimized between 0.5-2 U/50 μL, do not exceed 2 U/50 μL. In order to prevent polymerase from degrading primers due to 3 '→5' exonuclide activity, it is suggested to add polymerase to the reaction system in the last step.

5)Mg2+ final concentration: The final concentration of the system is 2 mM. Please ask our company to provide buffer with low Mg2+ concentration or without Mg2+ if you have special needs.

2. Recommended reaction procedure

Table 2. PCR amplification reaction procedure

[Notes]：

1)Initial denaturation temperature and time: 98℃ is recommended. The recommended time is 30 sec for simple templates such as plasmid DNA, 1 min for libraries, 3 min for complex templates such as cDNA and genomic DNA, and 5-10 min for high GC content templates.

2)Annealing temperature and time: 60℃ is recommended, or temperature gradient can be set up to find the optimal temperature for primer annealing according to needs. The recommended annealing time is 20 sec and can be adjusted within 10-30 sec. Too long annealing time may lead to dispersion of the amplified products on the gel.

3)Extension temperature and time: 72℃ is recommended. The extension time is adjusted according to actual needs.