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PCR Efficiency Formula:
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PCR efficiency refers to the rate at which DNA is amplified during each cycle of the polymerase chain reaction. It is calculated from the slope of the standard curve and indicates how effectively the target DNA is being copied.
The calculator uses the PCR efficiency formula:
Where:
Explanation: The efficiency is derived from the slope of the standard curve plot, where the slope is obtained from plotting the log of the starting quantity against the cycle threshold (Ct) values.
Details: Accurate PCR efficiency calculation is crucial for validating qPCR experiments, ensuring reliable quantification of gene expression, and maintaining the accuracy of relative quantification methods.
Tips: Enter the slope value obtained from your standard curve. The slope should be a non-zero value. Ideal PCR efficiency is typically between 90-110% (E = 0.9-1.1).
Q1: What is considered good PCR efficiency?
A: Good PCR efficiency typically ranges from 90% to 110% (E = 0.9-1.1). Efficiency outside this range may indicate issues with the reaction.
Q2: How is the slope obtained?
A: The slope is derived from a standard curve plot of log starting quantity versus cycle threshold (Ct) values from serial dilutions of a known template.
Q3: Why is PCR efficiency important?
A: PCR efficiency affects the accuracy of quantification. Inefficient reactions lead to underestimation of target quantity, while overly efficient reactions may cause overestimation.
Q4: What factors affect PCR efficiency?
A: Primer design, template quality, reaction conditions, enzyme performance, and inhibitor presence can all affect PCR efficiency.
Q5: How often should efficiency be calculated?
A: Efficiency should be calculated for each primer set and validated whenever reaction conditions change or when troubleshooting qPCR experiments.