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Vial / Bottle	Cap	Label	Function / Description	Content
1	colorless	LightCycler® Color Compensation Set, Blank	Standard buffer excluding fluorophores. To determine the standard buffer fluorescence.	1 vial, 100 μl
2	green	LightCycler® Color Compensation Set, Fluorescein Calibrator	Standard oligonucleotide, fluorescein-labeled at the 3' end, [0.3 μM]. To determine the crosstalk from the fluorescein channel 530 into channels 640 and 705.	1 vial, 100 μl
3	red	LightCycler® Color Compensation Set, LightCycler® Red 640 Calibrator	Standard oligonucleotide, LightCycler® Red 640-labeled at the 5' end, [1 μM]. To determine the crosstalk from the LightCycler® Red 640 channel 640 into channels 530 and 705.	1 vial, 100 μl
4	purple	LightCycler® Color Compensation Set, Cy5.5 Calibrator	Standard oligonucleotide, Cy5.5-labeled at the 5' end, [1 μM]. To determine the crosstalk from Cy5.5 channel 705  into channels 530 and 640.	1 vial, 100 μl

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Procedure	Assay Time [min]
Setup of the Calibration run	5
LightCycler® Carousel-Based System Calibration run	40
Total Assay Time	45

", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "

• Nuclease-free, aerosol-resistant pipette tips
• Pipettes with disposable, positive-displacement tips

• LightCycler^® Carousel-Based System*
• LightCycler^® Capillaries*
• Standard benchtop microcentrifuge containing a rotor for 2 ml reaction tubes

or

• LC Carousel Centrifuge 2.0* for use with the LightCycler^® 2.0 Sample Carousel (optional)

", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" }, { "Language": "en", "Value": "Ready-to-use solutions for the generation of color compensation files on the LightCycler^® Carousel-Based System Instruments", "Country": "XG", "Code": "Product Characteristics", "Name": "Product Characteristics" }, { "Language": "en", "Value": "Ready-to-use solutions for the generation of color compensation files for the LightCycler^® Carousel-Based System.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "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.", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "This is an excerpt from a Biochemica Help Corner article about Color Compensation. Please refer toBiochemica 2009, No. 2, page 27 for the full article.

What should be used for Color Compensation?

It is recommended to use fluorescent probes from the same batch/lot that are used in the multicolor experiment. The LightCycler^® Color Compensation Set (Cat. No. 12 158 850 001) contains ready-to-use solutions: however, the HybProbe probes contained in this kit will compensate only the 530 nm, 640 nm and 705 nm channels.
For TaqMan^® Probes or Molecular Beacons, it is recommended to set up an actual PCR with the specific primers, probes and template in the capillary (please see the LightCycler^® Technical Note No. 21/2007, \"Hydrolysis Probe Assays with the LightCycler^® System\").


How is a Color Compensation run performed?

A capillary dedicated to each dye and one for a blank (everything except the dye) is all that is required to perform a color compensation calibration run. The concentrations of the dyes should be similar to those actually used in the PCR.
If using a parameter-specific kit from Roche Applied Science, the LightCycler^® Color Compensation Set is recommended, which contains ready-to-use solutions. Otherwise we recommend using the probes that will be used in the assay, by setting up reactions using an appropriate PCR master mix, the probe and the volume adjusted with PCR-grade water.

A color compensation run is simply a melt curve; however, it is advisable to also run a PCR program to see how the probes behave during amplification. This enables a true assessment of any probe/signal degradation that may occur during multiple heating/cooling cycles of the PCR cycling, which could potentially affect the overall fluorescent signal.

A Color Compensation Set Macro is provided in the LightCycler^® Software 4.1. Alternatively a color compensation run can be performed using the same program settings as the multicolor experiment, including a melting program with the following segments:
Segment 1: +95°C for 0 sec at +20°C/sec ramp rate
Segment 2: +40°C for 30 sec at +20°C/sec ramp rate
Segment 3: +95°C for 0 sec at +0.1°C/sec ramp rate, with continuous acquisition

Once the LightCycler^® Instrument has been programmed and the maximum seek position (number of capillaries) has been selected in the Run key of the LightCycler^® Software 4.1, navigate to the Sample Editor key. In the Analysis Mode field, select Color Compensation.

In the Color Comp tab (shown after Color Compensation is selected), enter sample information and select the appropriate channel for each capillary. Select 'Water' for the first capillary (which should contain everything except the dye), then the appropriate channel from: 530, 560, 610, 640, 670, or 705 for each additional capillary. Using values that are not listed above will lead to errors.
Always place the 'Water' capillary containing everything except the dye (the blank) in the first position of the carousel. The PCR buffer (including Mg concentration) in the 'Water' capillary should be the same as that used in the assay to which the color compensation data will be applied.

Place the capillaries for the color compensation into the carousel from lowest to highest wavelength. For example:
Capillary 1 - Blank (everything except the dye)
Capillary 2 - Fluorescein
Capillary 3 - LightCycler^® Red 640
Capillary 4 - Cy5.5


How is a Color Compensation file/key created?

After the color compensation calibration run is complete, select Color Compensation from the Analysis menu. Ensure that the Color Compensation Analysis is open and saved, then click on Save CC Object on the analysis window toolbar. Save the Color Compensation key in the CCC folder, which is under the Special Data folder. Enter a name, then click OK.


When and how is Color Compensation applied?

Color Compensation can be applied wither before the multicolor experiment is run, or before the data analysis, after the run.


How is Color Compensation applied to macros?

When an Experiment Kit Macro is created, one of the following options may be selected:

• Auto select color compensation - the software will search for the last color compensation data set that matches the current Instrument ID and the color compensation ID, if entered on the Capillary View tab of the Sample Editor.
• Current color compensation choices - the color compensation data for the current experiment will be included in the kit macro.

", "Country": "XG", "Code": "Protocols - Help Corner", "Name": "Protocols - Help Corner" }, { "Language": "en", "Value": "This is an excerpt from a Biochemica Help Corner article about Color Compensation. Please refer to Biochemica 2009, No. 2, page 27 for the full article.

How do Color Compensated data look?

After a CC key has been generated in the LightCycler^® Software 4.1, both raw data and compensated data will be shown in the Color Compensation Analysis. To ensure that the color compensation has been created correctly, select the Channel button to look at the compensated data for each channel. The selected channel's fluorescence should be higher than that of the other channels and the fluorescence for all channels should always be higher than for the blank (Capillary 1). For example, with the 530 channel selected, usually the 530 nm fluorescence will decrease as the temperature increases; the other channels can be found at the bottom of the graph (this can be seen in Figure 1a of theHelpCorner article of Biochemica No. 2 2009). In the raw data of the 640 and 705 channels (Figures 1b and 1c of theHelpCorner article of Biochemica No. 2 2009), crosstalk of the 530 channel will be seen, but in the compensated data, only the dominant channel will show a curve and the other channels can be found at the bottom of the graph. The data for the blank capillary is seen at the bottom of each graph. If the lines of the fluorophores cross or come close to the blank, perform the Color Compensation calibration run again using fresh probes and reagents.


How often should Color Compensation be performed?

Color Compensation should be performed at least every 6 months, preferably every time a new lot of probes is used/reordered. If the multicolor assay will be performed on multiple LightCycler^® Instruments, the Color Compensation calibration run must be performed on every instrument. Color Compensation is instrument-specific. A new Color Compensation file should also be generated when the software is updated, or after the Instrument is repaired.", "Country": "XG", "Code": "Troubleshooting - Help Corner", "Name": "Troubleshooting - Help Corner" }, { "Language": "en", "Value": "This is an excerpt from a Biochemica Help Corner article about Color Compensation. Please refer to Biochemica 2009, No. 2, page 27 for the full article.

Color Compensation

The LightCycler^® Carousel-Based System is able to simultaneously detect and analyze more than one color in each capillary, thus there is a necessity for color compensation when performing dual and multicolor applications.


What is Color Compensation?

Fluorescent dyes emit over a broad range of wavelengths, thus there is overlap of emission spectra, or crosstalk, which can lead to data that can not be interpreted (by any software). Color compensation corrects for the spectral overlap and is used to subtract crosstalk of fluorescent signals outside the dominant channel.


Why is Color Compensation required?

Color Compensation is necessary only when dual and/or multicolor assays are run in a single capillary. It may not be required for all multicolor assays, if there is good separation of the spectral emissions of the fluorescent dyes (e.g., a HybProbe probe labeled with LightCycler^® Red 610 may not have significant spectral crosstalk with a HybProbe probe labeled with Cy5.5 {705}).


What are the factors that influence Color Compensation?

The following factors can influence the degree of crosstalk:

• temperature
• coupling chemistry of the fluorescent dyes
• properties of the optical system

To compensate for temperature, a color compensation calibration run must include a temperature gradient step (+40 to +95°C), in which data from all fluorescent dyes are continuously measured in all channels.
To minimize the effects of coupling chemistry, ensure that labeled probes are of high quality.
To compensate for the properties of the optical system, generate and store an instrument-specific color compensation file for all possible fluorophores that will be used. Optical systems will always show minor variations from instrument to instrument; thus it is important to generate an instrument-specific color compensation file.", "Country": "XG", "Code": "Background Information - Help Corner", "Name": "Background Information - Help Corner" } ] } } ] }