1. What is QPCR Amplification Factor?

The QPCR Amplification Factor (AF) represents the efficiency of DNA amplification in quantitative PCR. It's calculated from the slope of the standard curve and indicates how much product is amplified in each PCR cycle.

2. How Does the Calculator Work?

The calculator uses the amplification factor equation:

Where:

• \( AF \) — Amplification factor
• \( S \) — Slope of the standard curve

Explanation: The equation converts the standard curve slope into an amplification efficiency value between 1 and 2 (where 2 represents perfect doubling each cycle).

3. Importance of Amplification Factor

Details: The amplification factor is crucial for assessing PCR efficiency, validating experiments, and ensuring accurate quantification of target DNA.

4. Using the Calculator

Tips: Enter the slope value from your standard curve. The slope should be negative (typically between -3.1 and -3.6 for efficient reactions).

5. Frequently Asked Questions (FAQ)

Q1: What is an ideal amplification factor?
A: An ideal AF is 2.0, representing 100% efficiency (perfect doubling each cycle). Values between 1.8-2.1 are generally acceptable.

Q2: How do I get the slope value?
A: The slope is obtained from the standard curve plot of Ct values versus log input quantity in your qPCR software.

Q3: What does AF < 1.8 indicate?
A: AF below 1.8 suggests suboptimal PCR efficiency, possibly due to inhibitors, poor primer design, or suboptimal reaction conditions.

Q4: Can I use this for absolute quantification?
A: Yes, but you need to include proper standards and controls in your experiment.

Q5: How does this relate to efficiency percentage?
A: Efficiency % = (AF - 1) × 100. An AF of 2.0 equals 100% efficiency.