Storage Condition

Store at −30~−15℃ and protect from light. Transport at ≤0℃.

Components

Description

This universal premix is optimized for qPCR detection using the SYBR Green I fluorescence method. It contains hot-start Taq DNA Polymerase, dNTPs, reaction buffer, PCR enhancer, and universal corrective dyes compatible with all major qPCR instruments (including ROX-dependent systems). Only DNA template and primers need to be added.

The premix reduces setup time and contamination risk, while ensuring high efficiency, specificity, and consistent amplification across a wide range of templates.

Protocol

1) Notes

• Avoid strong light exposure to protect the dye.
• Mix gently by inversion before use; do not vortex.
• Contains universal corrective dyes for all qPCR instruments.

2) Reaction System (20 μl)

• Universal SYBR Green qPCR Mix: 10 μl (1×)
• Forward Primer (10 μM): 0.4 μl (0.2 μM final)
• Reverse Primer (10 μM): 0.4 μl (0.2 μM final)
• DNA Template: x μl (10–200 ng/20 μl)
• Nuclease-Free Water: to 20 μl

Primer concentration typically 0.2 μM; adjust between 0.1–1 μM. Template ≤10% reaction volume.

3) qPCR Programs

2-step PCR

• Pre-denaturation: 95℃ 30 s
• Denaturation: 95℃ 10 s
• Annealing & Extension: 60℃ 30 s
• Melting curve: per instrument

3-step PCR

• Pre-denaturation: 95℃ 30 s
• Denaturation: 95℃ 10 s
• Annealing: 55–65℃ 10 s
• Extension: 72℃ 30 s
• Melting curve: per instrument

Optimizations

• Primer concentration: lower = higher specificity, higher = higher efficiency.
• Primer design: 80–200 bp target length, 18–25 bp primers, Tm 58–62℃, GC 40–60%.
• For high specificity: 2-step PCR or higher annealing temperature.
• For higher efficiency: 3-step PCR or longer extension time.

FAQ & Troubleshooting

Unsmoothed or weak amplification curves

• Check dye integrity, use high-quality qPCR consumables.

No amplification curve

• Increase cycles to 40.
• Ensure proper signal acquisition settings.
• Check primer quality and template concentration.

Nonspecific amplifications

• Re-design primers or adjust primer concentrations.
• Analyze melt curves to confirm product specificity.

Poor repeatability

• Use accurate pipettes and proper technique.
• Increase reaction volume to reduce error.
• Verify instrument calibration.