1. What is qPCR Primer Efficiency?

Primer efficiency in quantitative PCR (qPCR) refers to the ability of primers to amplify the target sequence during each PCR cycle. An efficiency of 1 (or 100%) means the amount of product doubles with each cycle.

2. How Does the Calculator Work?

The calculator uses the standard curve slope efficiency equation:

Where:

• \( E \) — Primer efficiency (dimensionless)
• \( slope \) — Slope of the standard curve (Ct vs log(dilution))

Explanation: The slope of the standard curve relates to the amplification efficiency. An ideal slope of -3.32 corresponds to 100% efficiency.

3. Importance of Primer Efficiency

Details: Knowing primer efficiency is crucial for accurate quantification in qPCR experiments. Efficiency affects the calculation of relative gene expression and absolute quantification.

4. Using the Calculator

Tips: Enter the slope value from your standard curve (typically between -3.0 and -3.6). The calculator will compute the corresponding efficiency.

5. Frequently Asked Questions (FAQ)

Q1: What is an acceptable primer efficiency?
A: Typically 90-110% (0.9-1.1) is considered acceptable, with 95-105% being ideal.

Q2: How do I obtain the slope value?
A: Generate a standard curve by running serial dilutions of your template and plot Ct values against the log of template concentration.

Q3: What does a slope of -3.32 mean?
A: This indicates 100% efficiency, where the PCR product exactly doubles each cycle.

Q4: Why might my efficiency be outside the ideal range?
A: Poor primer design, PCR inhibitors, suboptimal reaction conditions, or template issues can affect efficiency.

Q5: Can I use this for absolute quantification?
A: Yes, but you must account for efficiency in your calculations for accurate results.