RT-PCR FAQ

Q: What are the required materials to perform Real-Time PCR?

A: SmartCycler® & software, computer configured for SC, PCR tubes, primers, probe or intercalating dye, dNTP's, MgCl2, DNA or cDNA, polymerase, water, centrifuge, pipet and pipet tips, buffers. SmartMix and OmniMix are available for convenience which contain all the reagents to perform a PCR reaction, except the primers and probes, in a SmartBead format.

Q: How do I design primers and probes?

A: Information regarding primer and probe design may be found in SmartNote 6.1 Designing Real-Time Assays on the SmartCycler®II System.

Q: How do I optimize my PCR reaction for Real-Time PCR on the SmartCycler®System?

A: Information regarding the design of Real-Time PCR assays may be found in SmartNote 6.1 Designing Real-Time Assays on the SmartCycler®II System.

Information regarding optimizing and analyzing Real-Time Assays on the SmartCycler® System may be found in SmartNote 6.2 Optimizing and Analyzing Real-Time Assays on the SmartCycler® II System.

Q: Which fluorophores should I label my probes with?

A: Information regarding fluorophores may be found in SmartNote 6.3 Dye-Quencher Considerations for the SmartCycler® II System.

Q: Can I run the product on a gel?

A: Yes, you can run the product on a gel by simply using a gel loading pipette tip to remove the contents from the SmartCycler® reaction tube.

Q: Why are there bands of larger DNA than the PCR product on my gels?

A: Possible cancatamers, which is when DNA self anneal to one another and start extending.

Q: Is it possible to perform a multiplex assay in the SmartCycler® II system?

A: Yes, the SmartCycler® II System contains the capability to detect up to 4 targets within the same tube. More targets may be detectable by using the Advance to Next Stage feature in the software. Please refer to SmartNote Advance to Next Stage.

Q: I have a positive growth curve in my negative control when I use an intercalating dye. What is this?

A: This may be a result of non-specific amplification. Please refer to SmartNote 6.1 Designing Real-Time Assays on the SmartCycler® II System.

This may be a result of primer-dimer generation. To minimize primer-dimer formation, please follow these guidelines:

• Use a hot-start enzyme to minimize extension of non-specific binding of primers during the reaction preparation.
• Determine the minimum amount of time for each step in the thermal cycling protocol and avoid excessive hold times.
• Use the lowest concentration of primer necessary for efficient amplification.
• Use the highest annealing temperature possible.
• Use the minimum number of cycles.
• Use the lowest concentration of MgCl2 concentration.
• Design primers with the highest Tm possible.
• Design primers that do not contain homologous sequences.
• Avoid C's and G's at the 3'end of the primers.