Real-time qPCR is a PCR method used to amplify and simultaneously quantify target DNA molecules. Two methods are frequently used for qPCR: double-strand DNA-binding dyes (e.g., SYBR® Green I) or fluorescent reporter probes (e.g., TaqMan®). In both cases, fluorescence signals are detected during the exponential phase.
Q: What is the function of Passive Reference Dye and how to use it?
A: They are reference fluorescent dyes used to normalize pipetting errors compatible with different instruments. Their concentration is provided at 50 ×, and 0.4 μl is needed for 20 µl reaction.
Q: Why do CT values appear too late?
A: It may be because the amplification efficiency is low due to the non-optimal reaction conditions. The degradation of PCR reaction components or too big size of PCR products (typically PCR products of 80-250 bp are required) could also cause late CT.
Q: Does Mg2+ concentration need to be adjusted in qPCR SuperMix?
A: Generally, it doesn’t need adjustment, but when the PCR results are not optimal or other adjustments cannot lead to significant improvement, amplification results can be improved by adjusting Mg2+ concentration.
Q: Why is the amplification curve abnormal?
A: The baseline setting is incorrect. High concentration of templates can cause low CT value, i.e. the baseline range becomes smaller, resulting in an incorrect reading of fluorescent values by the instrument.
Q: Why is the repeatability poor?
A:Operative errors.Low template concentration can lead to poor repeatability. It is recommended to increase template concentration or decrease the dilution fold.Temperatures between different wells of the instrument are not consistent.
Q:NTC is not zero, and how to optimize it?
A:Increase the annealing temperature.Reduce the working concentration of the primer. If necessary, the concentration can be reduced to 60 nM.It is recommended to operate on ice.Redesign the primer. If necessary, design multiple primers and choose the best one