1. What is QPCR Amplification Efficiency?

Amplification efficiency (E) in qPCR represents how efficiently the target DNA is amplified during each cycle of the PCR reaction. An efficiency of 100% means the amount of product doubles with each cycle.

2. How Does the Calculator Work?

The calculator uses the equation:

Where:

• \( E \) — Amplification efficiency (converted to percentage)
• \( S \) — Slope of the standard curve

Explanation: The slope of the standard curve (plot of Cq vs log input DNA) is used to calculate the amplification efficiency.

3. Importance of Amplification Efficiency

Details: Knowing the amplification efficiency is crucial for accurate quantification in qPCR experiments. Ideal efficiency is between 90-110%.

4. Using the Calculator

Tips: Enter the slope value obtained from your standard curve. The slope should be negative (typically between -3.1 to -3.6 for ideal efficiency).

5. Frequently Asked Questions (FAQ)

Q1: What is the ideal amplification efficiency?
A: The ideal efficiency is 100% (E = 1.0), corresponding to perfect doubling of product each cycle. Acceptable range is 90-110%.

Q2: What slope corresponds to 100% efficiency?
A: A slope of -3.32 corresponds to 100% efficiency. This is the theoretical ideal.

Q3: Why might efficiency be below 90%?
A: Low efficiency can result from poor primer design, PCR inhibitors, suboptimal reaction conditions, or template degradation.

Q4: Why might efficiency be above 110%?
A: High efficiency may indicate primer-dimer formation, contamination, or non-specific amplification.

Q5: How can I improve my qPCR efficiency?
A: Optimize primer design, annealing temperature, Mg2+ concentration, and ensure high-quality template DNA.