PrimeQ Real time PCR Filter Selection
When performing experiments requiring detection of more than one fluorophore, it is highly desirable for minimal overlap in signal from each dye. Whilst the choice of fluorophore used is important, the design of the optics for sample excitation and emission detection is crucial to obtain a sensitive and specific assay. The most specific method of exciting and detecting multiple dyes is to use a series of tailored excitation and emission filters. These ensure that only light of the correct wavelength reaches the dye to be excited and only emitted light of the correct wavelength reaches the detector.
FC02, FC03, FC04 and FC05 are supplied as standard with Techne PrimeQ and are optimised for use with FAM™, HEX™, ROX™ and Cy®5. For multiplexing FAM™ with alternative dyes which are detected with the FC03 filter, we recommend the use of FC01 which has lower excitation and emission wavelengths and a slightly wider bandwidth.
Crosstalk can occur when the emission of one fluorophore is detected through the filter combination of another fluorophore. Using specifically designed filters minimises or eliminates this effect and removes the requirement for complex colour compensation algorithms.
Using a combination of excitation and emission filters to eliminate crosstalk between dyes provides confidence in knowing that a signal has been generated by a specific probe without contribution from any other possible reactions in the same well. In addition, the fluorescent data does not have to be manipulated in any way in order to subtract any contribution to the signal by other dyes. This means that the results obtained are exactly what the detector sees i.e. the raw data, whether performing multiplex or single dye experiments.
Read the full application note at:
contact camlab for a demo or quotation of the Techne PrimeQ Real Time PCR