1. What is Amplification Efficiency?

Amplification efficiency (E) in qPCR represents how effectively a target sequence is amplified during each cycle of the PCR reaction. Ideal efficiency is 100%, meaning the amount of product doubles with each cycle.

2. How Does the Calculator Work?

The calculator uses the efficiency formula:

Where:

• \( E \) — Amplification efficiency (typically expressed as percentage)
• \( S \) — Slope of the standard curve

Explanation: The formula converts the standard curve slope into an efficiency value that indicates how close the reaction is to the ideal doubling of product each cycle.

3. Importance of Efficiency Calculation

Details: Knowing your qPCR efficiency is crucial for accurate quantification of target nucleic acids and for comparing expression levels between different samples.

4. Using the Calculator

Tips: Enter the slope value from your standard curve plot. The slope is typically between -3.1 and -3.6 for good qPCR assays.

5. Frequently Asked Questions (FAQ)

Q1: What is the ideal efficiency value?
A: 100% efficiency is ideal, corresponding to perfect doubling each cycle. Acceptable range is typically 90-110%.

Q2: What slope corresponds to 100% efficiency?
A: A slope of -3.32 corresponds to 100% efficiency (10^(-1/-3.32)-1 = 1.0 or 100%).

Q3: What causes poor efficiency?
A: Common causes include poor primer design, PCR inhibitors, suboptimal reaction conditions, or template issues.

Q4: How often should efficiency be checked?
A: Efficiency should be validated for each primer pair and checked periodically, especially when using new reagent lots.

Q5: Can I use this for absolute quantification?
A: Yes, but you need a proper standard curve and validated efficiency for accurate absolute quantification.