SYBR Green qPCR
SYBR dye fluorescence rises as DNA doubles each cycle; the cycle where it crosses a threshold (Ct) compares starting amounts. Halving the template delays Ct by about one cycle.
Key points
- Primer efficiency 90–110% with a single melt peak
- Three technical replicates and a no-template control
- Ct between 15 and 32 is the comfortable range
Materials
- 2× SYBR Green master mix
- Forward and reverse primers (10 µM each)
- cDNA
- Nuclease-free water
- qPCR plate and optical seal
- qPCR instrument
Steps
- 💡 The initial 95 °C time depends on the enzyme in your master mix.
Calculations
SYBR master mix (20 µL reaction, plus 2 µL cDNA)
🔬 Variable explorer — what changes if…?
The skeleton stays the same, but concentrations, times and reagents change between experiments. Click an item to see what it affects and compare two conditions (A and B) side by side.
cDNA amount (dilution)
What it isTemplate per well. Halving it delays Ct by about 1 (at 100% efficiency).
More diluteToo dilute: Ct above 35 with large replicate scatter.
More concentratedToo concentrated: RT reagents and inhibitors carry over and lower efficiency.
Typical choiceCt 15–32; dilute less for low-expressed genes.
Annealing temperature · primer design
What it isTemperature at which primers bind; must match primer Tm.
Lower · lessLower: primers bind elsewhere, giving non-specific products and primer dimers.
Higher · moreHigher: primers bind poorly, less product and later Ct.
Typical choiceUsually 60 °C for SYBR qPCR. Primers 18–25 nt, 40–60% GC, Tm within 3 °C of each other.
Primer concentration
What it isFinal primer concentration in the reaction.
Lower · lessLow (<100 nM): weak signal and late Ct.
Higher · moreHigh (>900 nM): primers pair with each other, adding a low-temperature melt peak (dimer).
Typical choice200–500 nM each.
Replicates
What it isTechnical replicates (same cDNA, several wells) catch pipetting error; biological replicates (independent samples) show real variation.
Lower · lessToo few: outliers cannot be spotted and statistics are impossible.
Typical choice3 technical and ≥3 biological replicates; statistics use biological replicates.
Reference gene
What it isThe gene used to correct for cDNA amount between samples.
If poorly chosenA reference that changes with treatment skews every result.
Typical choiceConfirm its Ct barely changes across conditions; use two or more if possible.
Troubleshooting
| Problem | Common cause | Fix |
|---|---|---|
| Amplification in NTC | Contamination or primer dimer | Distinguish by melt curve, fresh reagents, separate work areas |
| Two melt peaks | Non-specific product or dimer | Raise annealing temperature, lower primer concentration, redesign |
| Replicate Ct spread > 0.5 | Pipetting error, bubbles | Master-mix approach, spin plate, pipette ≥2 µL |
| Efficiency below 90% or above 110% | Inhibitors, dilution error, dimers | Repeat the standard curve with careful dilutions |
⚠ Safety SYBR dye binds DNA — handle with gloves.
This page summarises widely used general conditions. Reagent, kit and antibody manuals and your institution's safety rules take priority. Simulators marked "concept" are simplified models that show trends.
Analyse the results with free tools:
qPCR analysis (ΔΔCt, Pfaffl) → Primer efficiency standard curve → Primer resuspension →