Instrumentation & Calibration Flashcards

Which method best verifies temperature uniformity across a thermal cycler block?

Answer: A multi-probe temperature verification system placed in multiple wells

Multi-probe systems measure several well positions at the same time, which shows differences across the block.

In thermal cycler specifications, 'ramp rate' refers to:

Answer: The speed of temperature change between steps, in °C per second

Ramp rate is how fast the block heats or cools between setpoints, in °C/s.

What is the main purpose of a heated lid on a thermal cycler?

Answer: To prevent condensation and evaporation of the reaction onto the tube cap

Keeping the lid hotter than the block (often around 105°C) stops water from condensing under the cap, so the reaction volume stays constant.

A lab notices that the outer wells of a 96-well block consistently give later Cq values. What is the most likely instrument-related cause?

Answer: Edge effect from temperature non-uniformity at the block perimeter

Perimeter wells can heat and cool differently from center wells, which lowers amplification efficiency in a position-dependent way.

In many qPCR systems, the passive reference dye ROX is used to:

Answer: Normalize for non-PCR fluorescence variation such as small differences in well volume

ROX fluorescence stays constant during PCR, so dividing the reporter signal by it corrects for optical and volume differences between wells.

What is the purpose of a pure dye (spectral) calibration on a multicolor qPCR instrument?

Answer: To record each dye's spectral signature so overlapping signals can be separated

Pure dye spectra let the software separate the combined fluorescence into individual dye contributions.

A background calibration on a qPCR instrument is performed using:

Answer: A plate containing buffer or water without fluorescent dyes

Background calibration measures fluorescence from the plate, buffer, and optics so it can be subtracted from experimental data.