1. What is Primer Efficiency?

Primer efficiency is a measure of how effectively primers amplify their target sequence during PCR. It's expressed as a percentage, with 100% being the ideal efficiency where the amount of product doubles each cycle.

2. How Does the Calculator Work?

The calculator uses the primer efficiency equation:

Where:

• \( E \) — Primer efficiency (converted to percentage)
• \( slope \) — Slope of the standard curve from qPCR data

Explanation: The equation converts the slope of the standard curve (plotted as Ct vs log input DNA) into a measure of amplification efficiency.

3. Importance of Primer Efficiency

Details: Knowing primer efficiency is crucial for accurate quantification in qPCR experiments. Efficiency affects calculations of relative gene expression or absolute copy numbers.

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 primers (90-110% efficiency).

5. Frequently Asked Questions (FAQ)

Q1: What is an acceptable primer efficiency?
A: 90-110% is generally acceptable, with 95-105% being ideal. Below 90% or above 110% may indicate problems.

Q2: How do I get the slope value?
A: From your qPCR software's standard curve analysis. Plot Ct values against log10 of the template concentration.

Q3: Why is my efficiency over 100%?
A: This may indicate PCR inhibition in diluted samples, pipetting errors, or primer-dimer formation.

Q4: What if my efficiency is low?
A: Consider redesigning primers, optimizing annealing temperature, or checking for PCR inhibitors.

Q5: Can I use this for SYBR Green and probe-based assays?
A: Yes, the calculation works for both, though probe assays often have slightly higher efficiencies.