SYBR Green is a DNA-binding dye that fluoresces only when bound to double-stranded DNA. This characteristic makes it ideal for real-time detection of PCR products during each cycle of amplification. Its universality makes it an excellent choice for researchers targeting a wide range of sequences without the need for custom probes.

1. Primer Design

• Length: 18–24 nucleotides
• GC Content: 40–60%
• Tm: 58–62°C, with both primers closely matched
• Avoid secondary structures and primer-dimers

Tools such as Primer-BLAST and OligoAnalyzer are highly recommended.

2. Template Quality and Quantity

• Use high-quality, DNAse/RNAse-free samples
• Normalize input across samples
• Avoid inhibitors like EDTA, phenol, or heparin

3. Annealing Temperature

• Run a temperature gradient PCR to determine optimal annealing
• Too low = non-specific amplification
• Too high = low yield

4. MgCl₂ Concentration

• Typical: 1.5–3 mM
• Influences polymerase activity and primer binding

5. Melt Curve Analysis

Critical for SYBR Green validation:

• Single sharp peak = specific product
• Multiple peaks or broad peaks = non-specific products or primer-dimers

• No Template Control (NTC): Detect contamination or primer-dimers
• No Reverse Transcriptase Control (NRT): For RNA templates, confirms no genomic DNA contamination
• Positive Control: Ensures assay components are functioning

• Use software such as Bio-Rad CFX Manager or Thermo Fisher QuantStudio for analysis.
• Normalize against housekeeping genes (e.g., GAPDH, ACTB).
• For relative quantification, apply the 2^−ΔΔCq method.

At every stage of your qPCR experiment—from sample prep to final analysis—Gentaur supports your research with rigorously tested products and protocols.

🧬 Gentaur Offers:

• Validated SYBR Green kits for human, microbial, and viral diagnostics
• RNase-free water and buffers
• Plasmid and RNA extraction kits
• Custom qPCR primer services with melting curve verification

Gentaur’s products are manufactured under ISO-certified conditions and compatible with major qPCR platforms (Bio-Rad, Applied Biosystems, Roche, etc.).

📦 Order directly at gentaur.com or contact your local Gentaur distributor for expert guidance.