Your real-time PCR instrument may not be correctly calibrated

To ensure optimal performance of the thermal cycler or Sequence Detection System, it is strongly recommended that the thermal cycler is calibrated regularly. Calibration varies by instrument.

• Instrument calibration intervals

The different types of calibration are

• ROI calibration
• Background calibration
• Pure dye spectra calibration
• Instrument verification run

The ROI calibration allows the SDS software to map the position of the wells on the sample block so that, during instrument operation, the software can associate increases in fluorescence with specific wells of the reaction plates.

The background calibration measures the ambient fluorescence that is generated from background electrical signals, samples blocks, water inside consumables, and from the consumables themselves. This calibration enables the SDS software to eliminate background signal from the fluorescent samples, thus increasing the instrument’s precision.

The pure dye spectra calibration enables the instrument to distinguish the fluorescent dyes being used in the system. The SDS software uses the spectral data from a set of pure dye standards to process the raw spectral data it receives after each run.

The test verifies that the instrument can generate a standard curve and its ability to calculate the quantities of two unknowns. This test requires an RNAse P Verification Plate that contains pre-loaded reagents that create a standard curve with known copy numbers and two unknowns (also with known copy numbers).