Instrumentation & Calibration Flashcards

On camera-based qPCR instruments, a Region of Interest (ROI) calibration is used to:

Answer: Map the location of each well on the detector

ROI calibration tells the instrument which detector pixels correspond to each well.

In gravimetric pipette calibration, the dispensed volume is calculated by:

Answer: Weighing dispensed water and converting mass to volume with a density correction factor

The gravimetric method (ISO 8655) weighs dispensed water and applies a Z-factor for temperature and pressure to get volume.

During pipette calibration, the coefficient of variation (CV) of repeated dispenses measures:

Answer: Precision (random error)

CV reflects how closely repeated dispenses agree, which is precision or random error.

A temperature verification shows the denaturation step set at 95°C actually reaches only 93.5°C. What is the most likely effect on PCR?

Answer: Incomplete template denaturation and reduced amplification efficiency

If the denaturation temperature is too low, strands separate poorly, especially for GC-rich templates, and fewer templates are available each cycle.

After which event should a qPCR optical (dye/background) calibration typically be repeated?

Answer: Replacing the excitation lamp or optical components

Changes to the light source or optics change signal intensity and spectral response, so recalibration is needed.

Which symptom most strongly suggests an aging halogen excitation lamp in a qPCR instrument?

Answer: A gradual decrease in raw fluorescence across all wells over weeks

Lamp output falls over time, which lowers raw signal evenly across the whole plate.

Droplet digital PCR (ddPCR) instruments determine absolute target concentration by:

Answer: Applying Poisson statistics to the fraction of positive partitions

ddPCR counts positive and negative droplets and corrects for multiple copies per droplet with Poisson statistics, so no standard curve is needed.