Product Information

Product Description

      HRbio™ Hi-Fi High Fidelity DNA Polymerase is a PCR enzyme developed based on the KOD DNA polymerase of the hyperthermophilic original Thermococcus kodakaraensis KOD1 strain. The enzyme has 3'→5' exonuclease activity and has a higher fidelity than Taq DNA polymerase, which is 100 times higher. The addition of extension factors to the enzyme solution enables the enzyme to amplify long fragments, with an amplification speed of 15-30sec/kb, and the length of the amplified target fragment can be up to 18 kb. This product is equipped with an optimized buffer and added PCR enhancement components, making the enzyme suitable for the amplification of complex templates. The amplified product has a blunt end.

Product composition

Application

High-fidelity PCR; gene cloning; generation of sequencing templates; high GC content PCR; long fragment PCR (up to 1 8 kb); mutation high-throughput PCR

Activity Definition

Using activated salmon sperm DNA as template/primer, the activity of 1 U was defined as the amount of 10 nmol of total nucleotides taken up as acid-insoluble matter at 74°C for 30 min.

Transportation and storage methods

Transported with ice packs. Store at -20℃, valid for 1 year.

Precautions

1) For your safety and health, please wear a lab coat and disposable gloves when operating;

2) This product is for scientific research purposes only!

Recommended PCR reaction system (prepared on ice)

【Note】

1) Reagent use: Thoroughly thaw and mix before use.

2) Polymerase concentration: 1 U/50 μL is recommended, and can be optimized between 0.5-2 U/50 μL. Do not exceed 2 U/50 μL.

3) Addition of polymerase: To prevent the polymerase from degrading the primers due to 3'→5' exonuclease activity, it is recommended to add the polymerase to the reaction system in the last step.

4) Final concentration of Mg ²⁺ : The final concentration of the system is 1.5 mM. If there is a special need, 50 mM MgCl ₂ can be used , and the concentration can be increased by 0.2-0.5 mM.

5) High GC template: Adding DMSO to a final concentration of 3% to the system may facilitate amplification.

Recommended usage of different templates (50 μL reaction system):

PCR amplification program: There are three programs to choose from, and the two-step program is preferred.

Two-step method (preferred)

Three-step method (conventional procedure)

【Note】

1) Pre-denaturation temperature and time: Recommended temperature: 94°C, time: 2 min, 5-10 min for templates with high GC content.

2) Annealing temperature and time: It can be set according to the temperature of primer annealing. The recommended annealing time is 30 seconds, which can be adjusted within 10-30 seconds. Annealing time that is too long may cause the amplification product to be diffuse on the gel.

3) Extension temperature and time: Recommended temperature: 68°C. Time: 30 sec/kb. For complex templates, it can be extended to 60 sec/kb according to actual conditions.

4) Amplification product: Please store the PCR amplification product at -20℃ to prevent the enzyme from degrading the amplification product. The amplification product has a blunt end.