Thermal Cycling Parameters & Optimization Flashcards

Which cycling profile is typical for a standard two-step qPCR assay?

Answer: 95°C for 15 s, then 60°C for 60 s combined annealing/extension

Most qPCR master mixes use a short 95°C denaturation followed by a combined 60°C anneal/extend step.

After a SYBR Green qPCR run, a melt curve shows a single sharp peak. What does this suggest?

Answer: One specific amplification product

One product melts at one characteristic Tm, giving a single peak, while extra peaks point to nonspecific products or dimers.

In a two-step qPCR protocol, when is fluorescence usually measured?

Answer: At the end of the combined annealing/extension step

Measuring after annealing/extension captures the signal from newly made double-stranded product or cleaved probes in each cycle.

A qPCR standard curve has a slope of -3.32. What amplification efficiency does this mean?

Answer: About 100%

Efficiency = 10^(-1/slope) - 1, so a slope of -3.32 gives about 100%, meaning the product doubles each cycle.

In a one-step RT-PCR protocol, which step comes before the initial denaturation?

Answer: A reverse transcription incubation at about 42-55°C for 10-30 minutes

cDNA must first be made from RNA at the reverse transcriptase's best temperature, and the following high-temperature step also inactivates the RT.

For long-range PCR (more than 10 kb), which cycling change is usually recommended?

Answer: Use a 68°C extension with long extension times, often increased in later cycles

A lower extension temperature and longer times help long products finish while reducing template depurination.

A primer pair has Tm values of 54°C and 64°C. What is the best long-term fix?

Answer: Redesign the primers so their Tm values are within about 2-5°C

A large Tm mismatch means no single annealing temperature works well for both primers, so redesign is the reliable fix.