1. What is the Real-time PCR Copy Number Equation?

The Real-time PCR Copy Number equation estimates the relative quantity of DNA template in a PCR reaction based on the threshold cycle (Ct) and PCR efficiency (E). It provides a quantitative measure of the starting material in a PCR reaction.

2. How Does the Calculator Work?

The calculator uses the Copy Number equation:

Where:

• \( CN \) — Copy number (dimensionless)
• \( E \) — PCR efficiency (dimensionless, typically between 1-2)
• \( Ct \) — Threshold cycle (dimensionless)

Explanation: The equation shows the exponential relationship between the threshold cycle and the initial copy number, with PCR efficiency affecting the rate of amplification.

3. Importance of Copy Number Calculation

Details: Accurate copy number estimation is crucial for quantifying DNA templates, comparing gene expression levels, and determining viral loads in diagnostic applications.

4. Using the Calculator

Tips: Enter PCR efficiency (typically between 1.8-2.0 for optimal reactions) and threshold cycle value from your real-time PCR instrument. All values must be valid (efficiency > 0, Ct ≥ 0).

5. Frequently Asked Questions (FAQ)

Q1: What is a typical PCR efficiency value?
A: Ideal PCR efficiency is 2.0 (100% efficient), but values between 1.8-2.0 are generally acceptable.

Q2: How is threshold cycle (Ct) determined?
A: Ct is the cycle number at which the fluorescence exceeds a defined threshold, typically set in the exponential phase of amplification.

Q3: Can this be used for absolute quantification?
A: For absolute quantification, you need a standard curve with known quantities to relate Ct values to actual copy numbers.

Q4: What factors affect PCR efficiency?
A: Primer design, template quality, reaction conditions, and inhibitor presence can all affect efficiency.

Q5: How should I interpret the copy number result?
A: The result represents relative quantity. For meaningful interpretation, compare to controls or standards run in the same experiment.