Storage Condition

−20℃

Components

Description

The 2× S705 HiFi Master Mix (+Dye) is a ready-to-use premix containing S705 High-Fidelity DNA Polymerase, dNTPs, and optimized reaction buffer. Simply add template, primers, and Nuclease-Free Water to perform high-fidelity PCR amplification.

S705 High-Fidelity DNA Polymerase was obtained through directed evolution. Its fidelity is ~70-fold higher than Taq and 5× faster than Pfu polymerase. With enhancers, it supports fragments up to 20 kb from simple templates and 12 kb from complex genomic DNA. It also tolerates common PCR inhibitors.

Premix includes red and yellow tracking dyes, enabling direct gel loading. (Red ≈ 1500 bp, Yellow ≈ 10 bp in 1% agarose gel.)

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 (+Dye): 25 μl
• PCR Enhancer (optional): up to 10 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. Do not exceed recommended amounts to avoid inhibition. Touchdown PCR is advised.

2) Recommended Template Input

• Genomic DNA: 10–200 ng
• Plasmid/Viral DNA: 10 pg–50 ng
• cDNA: 1–5 μl (≤10% of reaction volume)
• Crude template: 1–5 μl (≤10% of reaction 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: Start Annealing 68℃, decrease 0.2℃/cycle.

Notice

• Do not use primers/templates with uracil or dUTP.
• S705 polymerase produces blunt ends. For T/A cloning, purify PCR product before A-tailing.
• Use high-quality template DNA for best yield.
• When programming thermocycler, select “Tube” mode. “Block” mode can cause ramp-rate issues with complex templates.

FAQ & Troubleshooting

Low yield / no amplification

• Optimize primer design.
• Adjust annealing temperature (use gradient).
• Increase primer concentration or extension time (30–60 s/kb).
• Increase cycle number (up to 40).
• Check template quality/amount.

Impurity / diffuse bands

• Increase annealing temperature or adjust primer concentration.
• Reduce cycle number (25–30).
• Use high-purity template DNA.