Instrumentation & Calibration Flashcards

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

Answer: 100%

Efficiency = 10^(-1/slope) - 1, so a slope of -3.32 gives about 100%.

Which standard curve slope falls OUTSIDE the commonly accepted 90-110% efficiency range?

Answer: -3.85

A slope of -3.85 corresponds to about 82% efficiency, which is below the acceptable range of roughly -3.58 to -3.10.

Where should the fluorescence threshold be set on a qPCR amplification plot?

Answer: Within the exponential phase, above background noise

A threshold in the exponential phase gives reproducible Cq values that reflect starting quantity.

Baseline cycles for qPCR analysis should be chosen so that they:

Answer: Cover cycles before any detectable amplification signal

The baseline should cover only background cycles, usually around 3-15, ending before the earliest amplification rises.

What is the primary instrumental advantage of a gradient thermal cycler?

Answer: It tests several annealing temperatures across the block in a single run

A gradient block holds different columns at different temperatures, so annealing conditions can be optimized in one run.

Temperature verification probes used for thermal cycler calibration should ideally be:

Answer: Traceable to a national standard such as NIST

Traceability to a national standard keeps calibration results accurate and defensible for audits.

Dust or residue inside thermal block wells primarily causes:

Answer: Poor heat transfer that causes well-specific temperature variability

Debris prevents good contact between tube and block, so samples in affected wells heat and cool unevenly.