For general laboratory use. Others LightCycler Color Compensation Set GLU LightCycler® Color Compensation Set 3.5.5.2.2.1 12158850001 LC Color Compensation Set LightCycler Color Compensation Set 04038377016719 Reagents, kits 1 set 4 vials5 calibration runs false Use the LightCycler® Color Compensation Set to generate color compensation data keys (LightCycler® Software 4.1). This is a prerequisite for performing dual-color experiments with both LightCycler® Red 640-labeled and Cy5.5-labeled HybProbe probes in a single capillary.The generated color compensation file/key can universally be used in all subsequent dual-color experiments on the specific LightCycler® Instrument to compensate for the raw fluorescence data either online during the run, or for data analysis after the run. en Ready-to-use solutions for the generation of color compensation files for the LightCycler® Carousel-Based System. en The LightCycler® Color Compensation Set is used to calibrate the LightCycler® Instrument to correct the spectral overlap of the fluorescence channels in dual-color applications that use LightCycler® Red 640-labeled and Cy5.5-labeled HybProbe probes. During a calibration run, the LightCycler® Instrument measures the fluorescence of each dye in all channels and generates an instrument-specific color compensation key. Later, the software automatically uses this color compensation file/key to reassign the fluorescence in each channel to the appropriate dye. The net result is detection of only one dye signal in each channel.The temperature profile used in a color compensation protocol always includes a Heating Step, a Cycling Step, a Temperature Gradient Step, and a Cooling Step. The cycling step mimics a typical PCR, including data acquisition. However, the data required for color compensation are taken from the temperature gradient step. In this step, after a brief denaturation (95°C), the protocol slowly increases the temperature, in increments of 0.2°C/second, from 40°C to 95°C, while continuously acquiring data. After the calibration run, the LightCycler® Software saves the data generated as a normal experiment. For these data to be used for color compensation, you must first convert the data into a .ccc key and save it again.The LightCycler® Carousel-Based System can detect two or more different sequences in one sample (e.g., both a target and an internal control). Such assays require two specific sets of HybProbe probes. HybProbe probes consist of two different short oligonucleotides that bind to an internal sequence of the amplified fragment during the annealing phase of the amplification cycle. One probe is labeled at the 5' end with a red fluorophore (LightCycler® Red 640 or Cy5.5) and in addition, is 3' phosphorylated to avoid extension. The other probe is labeled at the 3' end with fluorescein. Only after hybridization to the template DNA, the two probes come into close proximity, resulting in Fluorescence Resonance Energy Transfer (FRET) between the two fluorophores. During FRET, fluorescein, the donor fluorophore, is excited by the light source of the LightCycler® Instrument and part of the excitation energy is transferred to the red, acceptor fluorophore. The emitted fluorescence of the red fluorophore is then measured by the LightCycler® Instrument.Although the optical filters of each detection channel are optimized for different emission maxima, all fluorescent dyes currently available have emission spectra with long tails, leading to a spectral overlap. As depicted in Figure 1, the emission spectrum of the donor dye (fluorescein) slightly overlaps the emission spectrum of LightCycler® Red 640, which in turn, overlaps the Cy5.5 emission spectrum.Fig. 1: Emission spectra of fluorescent dyes.The emitted fluorescence intensities and spectra of fluorescent dyes are strongly temperature dependent. Therefore, the temperature chosen for data acquisition significantly influences the crosstalk. To enable crosstalk compensation in multicolor experiments at any temperature between 40°C and 95°C, the generation of a color compensation key includes a temperature gradient step with continuous data acquisition of the fluorescence measured in all three channels. en