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qPCR Master Mix FAQs
1. Q: What is the advantage of working with a probe system?
A: The probe system is always specific; only detect the gene of interest. It is also possible to distinguish between similar sequences with small differences like SNPs or mutations. In general, probe assays need less optimization than SYBR Green I assays
2. Q: Can I use a qSTAR SYBR Kit for a probe assay?
A: This is not possible because the buffer for qSTAR SYBR kits and the buffer for probe kits are different, and have been optimized for their specific assays
3. Q: Why do the qSTAR kits contain a hot-start Taq polymerase?
A: Polymerase activity during the reaction set-up causes non-specific amplification including primer-dimer formation. To avoid non-specific amplification the polymerase is only activated after heating at 95°C for 10 minutes
4. Q: Why do certain kits contain a ROX passive reference?
A: The emission recorded from ROX during the baseline cycles is used to normalize the emission recorded from the reporter (SYBR) in later cycles in some instruments. ROX compensates for small fluorescent fluctuations such as bubbles and well-to-well variations that may occur.
5. Q: Why are the ROX concentrations different?
A: The amount of the ROX passive reference dye needed varies depending on the instrument optics, our qSTAR SYBR kits have been optimized for these different instruments
6. Q: What is the difference between using ROX and using fluorescein?
A: Fluorescein is an alternative passive reference dye used just in the Bio-Rad instruments, the qSTAR SYBR & Fluorescein contains fluorescein premixed in the master mix at optimized concentrations.
7. Q: What is the difference between a one-step and a two-step real-time PCR reaction?
A: In a one-step reaction the reverse transcription reaction and the real-time PCR reaction are done in one tube, making this a closed tube assay, so contamination can be avoided. It saves pipeting steps and time, and is easy in handling, making it ideal for high throughput screening.