Storage Condition

−20℃

Components

Description

The 2× S705 HiFi Master Mix is a ready-to-use premix containing S705 High-Fidelity DNA Polymerase, dNTPs, and optimized buffer. Simply add template, primers, and water to start high-fidelity PCR.

S705 polymerase is engineered through directed evolution, offering ~70× fidelity of Taq and 5× faster speed than Pfu. With extension factors and enhancers, it enables long-fragment amplification up to 20 kb (simple templates) and 12 kb (genomic DNA), with high tolerance to PCR inhibitors.

Formulated for high specificity, yield, and robustness—even with GC-rich templates or crude extracts.

Applications

• PCR amplification from genomic DNA, cDNA, plasmids, or crude extracts
• High-fidelity cloning
• Long-fragment PCR

Protocol

1) Reaction System (50 μl)

• 2× S705 HiFi Master Mix: 25 μl
• 2.5× PCR Enhancer (optional): up to 20 μl^a
• Forward primer (10 μM): 1 μl
• Reverse primer (10 μM): 1 μl
• Template DNA: x μl
• ddH₂O: to 50 μl

^a For GC-rich templates (>60%), add enhancer and use Touchdown PCR.

2) Recommended Template Input

• Genomic DNA: 10–200 ng
• Plasmid/Viral DNA: 10 pg–50 ng
• cDNA: 1–5 μl (≤10% total volume)
• Crude template: 1–5 μl (≤10% total volume)

3) Cycling Programs (30–35 cycles)

Three-step PCR

• Initial Denaturation: 95℃ 3–5 min
• Denaturation: 95℃ 10 s
• Annealing: 55–72℃ 15 s
• Extension: 72℃ 30 s/kb
• Final Extension: 72℃ 5 min

Two-step PCR

• Initial Denaturation: 95℃ 3–5 min
• Denaturation: 95℃ 10 s
• Annealing & Extension: 65–68℃ 30 s/kb
• Final Extension: 72℃ 5 min

Touchdown PCR: Annealing starts at 68℃, decrease 0.2℃/cycle; 30–40 cycles.

Notice

• Do not use primers/templates containing uracil or dUTP.
• S705 polymerase produces blunt ends. For T/A cloning, purify PCR products before A-tailing.
• Use high-quality templates to improve yield.

FAQ & Troubleshooting

Low yield / no amplification

• Check primer design.
• Use gradient annealing to optimize Tm.
• Adjust primer concentration.
• Extend time: 30–60 s/kb.
• Increase cycles to 36–40.
• Verify template quality and input.

Impurity / diffuse bands

• Increase annealing temperature or adjust primers.
• Reduce cycles (25–30).
• Ensure template purity.