1. What is qPCR Efficiency?

qPCR efficiency (E) measures how effectively template DNA is amplified during each PCR cycle. Ideal efficiency is 100%, meaning the amount of product doubles with each cycle.

2. How Does the Calculator Work?

The calculator uses the qPCR efficiency formula:

Where:

• \( slope \) — Slope of the standard curve from your qPCR data
• \( E \) — PCR efficiency expressed as percentage

Explanation: The slope is determined by plotting the log of the template concentration against the Ct value. An ideal slope of -3.32 indicates 100% efficiency.

3. Importance of PCR Efficiency

Details: Accurate efficiency calculation is crucial for reliable quantification in qPCR experiments. Poor efficiency (typically <90% or >110%) indicates problems with primers, template quality, or reaction conditions.

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). The calculator will return the efficiency as a percentage.

5. Frequently Asked Questions (FAQ)

Q1: What is the ideal qPCR efficiency?
A: The ideal efficiency is 100%, which corresponds to a slope of -3.32. Acceptable range is typically 90-110%.

Q2: What causes poor PCR efficiency?
A: Common causes include poor primer design, PCR inhibitors, template degradation, suboptimal reaction conditions, or pipetting errors.

Q3: How can I improve my qPCR efficiency?
A: Redesign primers, optimize annealing temperature, check template quality, ensure proper reagent concentrations, and verify pipetting accuracy.

Q4: Should I use this efficiency value in my ΔΔCt calculations?
A: Yes, if your efficiency differs significantly from 100%, you should incorporate the efficiency value into your relative quantification calculations.

Q5: How often should I check my qPCR efficiency?
A: Efficiency should be checked with every new primer pair and periodically thereafter to ensure consistent performance.