High-Fidelity DNA Polymerase Kit

a. High-Fidelity Reaction buffer (5×)contains10 mMof Mg²⁺,and in the1×working solution, the final concentration is2 mM, so no additional addition is required for routine amplification; the separately provided25mMMgCl₂is only used for optimizing conditions in specific primer/template systems.

1.System Preparation

All operations should be performed on ice throughout the process. After thawing, thoroughly mix each component and promptly return it to -20°C storage after use.

a. The reaction volume can be adjusted between 10 and 50 μL. For volumes other than 25 μL, the reagents should be diluted proportionally. Reaction volumes greater than 50 μL are not recommended;

b. For most reactions, Mg²⁺the optimal final concentration is 1.5–2 mM. The system already contains a final concentration of 2 mM Mg²⁺; if necessary, the 25 mM MgCl₂provided in the kit can be used to incrementally adjust the Mg²⁺concentration in steps of 0.2–0.5 mM to determine the optimal concentration;

c. After preparation, gently tap the bottom of the tube to mix thoroughly and briefly centrifuge; avoid vigorous vortexing;

d. The optimal reaction concentration varies depending on the template; the following table shows the recommended template amounts for a 25 μL reaction:

2.Reaction program

a. Pre-denaturation conditions: For simple plasmids/λDNA, 95℃ for 2 minutes is sufficient; for complex genomic DNA, 95℃ for 3–5 minutes is recommended.

b. Denaturation conditions: 98℃ for 20 seconds is recommended. If the fragment is >10 kb, the denaturation temperature can be lowered to 92–94℃ for 15 seconds to reduce thermal damage.

c. Annealing temperature: In the High-Fidelity Reaction buffer system, primer annealing kinetics may be slightly altered under high-fidelity optimization. It is recommended to set the annealing time to 10–15 seconds. For initial experiments, a temperature gradient is recommended to determine the optimal annealing temperature.

d. Number of cycles: To maintain the highest fidelity, it is advised to limit the number of cycles to ≤25–35 cycles.

3.PCR procedure for high GC content and difficult templates

When the GC content of the template amplicon exceeds 60% or when the template has complex secondary structures, it is recommended to adjust the protocol as follows: on the basis of the standard reaction system, introduce a solvent additive—add DMSO—at a final concentration of 2%–8% in a 50 μL reaction (i.e., add 1–4 μL; it is recommended to explore using increments of 1% as a gradient).

(Note: Excessively high concentrations of the cosolvent may slightly reduce enzyme fidelity; it is recommended to use it only for challenging templates.)

a. Ice-bench operations: High-Fidelity DNA Polymerase is a hot-start enzyme system; although it allows for room-temperature reaction setup, performing operations on ice can yield exceptionally uniform experimental results.