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.

qPCR FAQs

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

Q: Do you have some recommand machine for qPCR?

A:

AZURE CIELO™ Real-Time PCR System, the machine can be used for In-vitro Diagnostics.

TransStart^® Green qPCR SuperMix UDG

TransStart^® Top Green qPCR SuperMix

TransStart^® Tip Green qPCR SuperMix

TransScript^® Green miRNA Two-Step qRT-PCR SuperMix

TransScript^® Green One-Step qRT-PCR SuperMix

TransScript^®  II Green One-Step qRT-PCR SuperMix

TransScript^® Probe One-Step qRT-PCR SuperMix

TransScript^®II Probe One-Step qRT-PCR SuperMix

PerfectStart^® II Probe qPCR SuperMix

PerfectStart^® Universal Green qPCR SuperMix