qPCR and qRT-PCR SuperMixes
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
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
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TransStart® Green qPCR SuperMix
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TransStart® Green qPCR SuperMix UDG
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TransStart® Top Green qPCR SuperMix
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TransStart® Tip Green qPCR SuperMix
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TransScript® Green Two-Step qRT-PCR SuperMix
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TransScript® Green miRNA Two-Step qRT-PCR SuperMix
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TransScript® II Green Two-Step qRT-PCR SuperMix
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TransScript® Green One-Step qRT-PCR SuperMix
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TransScript® II Green One-Step qRT-PCR SuperMix
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PerfectStart™ Green qPCR SuperMix
PCR, RT-PCR, qPCR, qRT-PCR
Restriction/Modifying Enzymes
DNA Molecular Weight Standards
Cloning and Mutagenesis System
Nucleic Acid Purification
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Gene Expression
Protein Analysis
Protein MW Standards
Cell Culture and Detection
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Residual DNA Detection
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