Real time PCR 2 Flashcards
What is the purpose of the Threshold value in Real Time PCR?
The Threshold value is used to compare two samples and determine the Ct value. It is set based on a baseline level of fluorescence and indicates the early exponential phase of amplification
How is the Threshold value determined?
The Threshold value is set at a level of fluorescence that is 10 standard deviations above the baseline, indicating the onset of amplification.
What does the Ct value represent in Real Time PCR?
The Ct value represents the cycle at which a sample reaches the Threshold level of fluorescence, allowing for comparison between samples.
What is the role of ROX in Real Time PCR?
ROX is a passive reference dye whose fluorescence levels are monitored throughout the reaction to normalize the fluorescence levels of the reporter molecule between different samples.
How does normalization occur in Real Time PCR?
Normalization is achieved by dividing the reporter fluorescence level by the ROX fluorescence level, ensuring that the relative fluorescence levels between samples are comparable.
What does the No template control indicate in Real Time PCR?
Represented by a flat line on the graph, ensures that the reagents are not contaminated, as its fluorescence should not increase during the PCR reaction.
How does the concept of a race analogy relate to Real Time PCR?
In the analogy, the finish line represents the Threshold value of fluorescence, and the time it takes for each sample to reach this threshold corresponds to its Ct value, allowing for comparison between samples.
