1. What is PCR Primer Efficiency?

PCR primer efficiency (E) measures how effectively primers amplify their target sequence during PCR. An efficiency of 1 (or 100%) means the amount of product doubles each cycle. Ideal efficiencies range between 90-110%.

2. How Does the Calculator Work?

The calculator uses the standard efficiency equation:

Where:

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

Explanation: The slope (S) is determined from a standard curve plot of Cq values versus log template quantity. The efficiency is then calculated from this slope.

3. Importance of Primer Efficiency

Details: Knowing primer efficiency is essential for accurate quantification in qPCR experiments. Poor efficiency can lead to inaccurate results when comparing gene expression levels.

4. Using the Calculator

Tips: Enter the slope value from your standard curve. Typical values range between -3.1 to -3.6, corresponding to efficiencies of 90-110%.

5. Frequently Asked Questions (FAQ)

Q1: What is an acceptable efficiency range?
A: 90-110% is generally acceptable, with 100% being ideal. Values outside this range may require primer redesign.

Q2: How is the slope determined?
A: The slope comes from plotting Cq values against the log of template concentration in a dilution series.

Q3: What causes poor efficiency?
A: Common causes include primer-dimers, secondary structures, suboptimal annealing temperatures, or inhibitor presence.

Q4: Can I use this for SYBR Green assays?
A: Yes, this calculation applies to both SYBR Green and probe-based assays.

Q5: How often should I check primer efficiency?
A: Efficiency should be validated for each new primer set and rechecked periodically or when changing reaction conditions.