Instrumentation & Calibration Flashcards

Most modern thermal cyclers heat and cool the block using:

Answer: Peltier (thermoelectric) elements

Peltier elements move heat in either direction depending on the direction of current flow, which allows fast, precise heating and cooling.

Why do many cycler protocols require the reaction volume to be entered in the software?

Answer: To let the sample-temperature algorithm estimate how far the sample lags behind the block

Sample temperature lags block temperature by an amount that depends on volume, and control algorithms use the volume to adjust timing and overshoot.

Using a low-profile plate in a block designed for standard-profile plates most likely causes:

Answer: Poor tube-to-block contact and inconsistent heating

Mismatched consumables do not seat properly in the wells, which reduces heat transfer and can cause evaporation.

What is an advantage of white-walled qPCR plates over clear plates?

Answer: Higher reflected signal and reduced well-to-well optical crosstalk

White walls reflect fluorescence toward the detector and block light from neighboring wells.

In instrument qualification, which stage confirms that the instrument performs consistently with the lab's actual assays under routine conditions?

Answer: Performance Qualification (PQ)

PQ shows reliable performance in real-world use, while IQ covers installation and OQ covers operation against specifications.

High-resolution melt (HRM) analysis places the greatest demand on which instrument capability?

Answer: Excellent thermal uniformity and dense fluorescence data collection during the melt

HRM detects tiny shifts in melt temperature, so it needs uniform temperatures across wells and very fine data steps.

On a UV spectrophotometer used to check PCR template, an A260 reading of 1.0 for double-stranded DNA corresponds to approximately:

Answer: 50 µg/mL

The standard conversion factor for dsDNA is about 50 µg/mL per A260 unit (40 for RNA and about 33 for ssDNA).