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qPCR Efficiency Calculator
PCR technology is widely used to aid in quantifying DNA because the amplification of the target sequence allows for greater sensitivity of detection than could otherwise be achieved. In an optimized reaction, the target quantity will approximately double during each amplification cycle. The efficiency of this reaction can be determined using a serial dilution of a positive control template (standard curve). The slope of the line of best fit drawn to the standard curve is used to determine reaction efficiency. The standard curve plots the log of starting template vs. PCR cycle number. A linear fit with a slope between approximately -3.1 and -3.6, equivalent to a calculated 90-110% reaction efficiency, is typically acceptable for most applications requiring accurate quantification.
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How to use this tool
1. Enter the data into the box on the right. Data can be directly copied from Excel or pasted as values in comma-separated, tab-separated, or space-separated formats. If the data is being entered manually, only place one value per line. The format should be as follows:
Place the cycle value into the first column. The subsequent columns (Data Set 2 and onwards) should hold the fluorescence values of the qPCR progression. Users can insert up to ten fluorescence variables per analysis. To add a new data set, press on the ‘+’ tab above the data entry area. Variables can be named by double clicking the tab but is optional.
| Cycle count | Fluorescence value | … |
| X1 | Y1 | … |
| X2 | Y2 | … |
| X3 | Y3 | … |
| X4 | Y4 | … |
| … | … | … |
Place the cycle value into the first column. The subsequent columns (Data Set 2 and onwards) should hold the fluorescence values of the qPCR progression. Users can insert up to ten fluorescence variables per analysis. To add a new data set, press on the ‘+’ tab above the data entry area. Variables can be named by double clicking the tab but is optional.
2. Verify your data is accurate in the table that appears.
3. Press the "Calculate qPCR efficiency" button to display results. Each dataset will generate an output in the form of a summary table comprising of hill coefficients, amplification factors, and qPCR efficiency percents.
Data Entry
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This online tool may be cited as follows
MLA | "Quest Graph™ qPCR Efficiency Calculator." AAT Bioquest, Inc., 21 Nov. 2024, https://www.aatbio.com/tools/qpcr-efficiency-calculator. | |
APA | AAT Bioquest, Inc. (2024, November 21). Quest Graph™ qPCR Efficiency Calculator. AAT Bioquest. https://www.aatbio.com/tools/qpcr-efficiency-calculator. | |
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