1. What is PCR Efficiency?

PCR efficiency refers to the rate at which PCR amplifies the target DNA sequence during each cycle of the reaction. An efficiency of 100% means the amount of product doubles with each cycle.

2. How Does the Calculator Work?

The calculator uses the PCR efficiency formula:

Where:

• \( slope \) — Slope from the standard curve plot (Ct vs log template quantity)

Explanation: The slope is derived from plotting the cycle threshold (Ct) values against the logarithm of the template concentration. The efficiency is then calculated from this slope.

3. Importance of PCR Efficiency

Details: Knowing PCR efficiency is crucial for accurate quantification in qPCR experiments. Ideal efficiency is between 90-110%. Efficiency outside this range may indicate problems with the assay.

4. Using the Calculator

Tips: Enter the slope value obtained from your standard curve. The slope should be negative (typically between -3.1 to -3.6 for good efficiency).

5. Frequently Asked Questions (FAQ)

Q1: What is the ideal PCR efficiency?
A: The ideal efficiency is 100%, meaning the DNA doubles each cycle. In practice, 90-110% is generally acceptable.

Q2: What does low efficiency indicate?
A: Low efficiency (<90%) may indicate PCR inhibition, poor primer design, or suboptimal reaction conditions.

Q3: What does high efficiency indicate?
A: Efficiency >110% may indicate primer-dimer formation or contamination.

Q4: How is the slope determined?
A: The slope is obtained from the standard curve plot of Ct values versus the log of template concentration.

Q5: Can I use this for absolute quantification?
A: Yes, but you need a proper standard curve along with efficiency calculations for accurate absolute quantification.