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LightMix® Universal Color Compensation Hexaplex

Color Compensation Reagents for LightCycler® 480 / 480 II Instruments and cobas z 480 Analyzer.
TIB MOLBIOL order number: 40-0320-00

For life science research only. Not for use in diagnostic procedures. For in vitro use only.

Product No. Pack Size
06296971001 96 reactions

To order these products, please contact your Roche order management team.

The emission spectra of the fluorophores used with the Real-Time-PCR Instruments overlap, causing fluorescence signals to be recorded in the adjacent channels when used simultaneously. The Color Compensation (CC) is a tool of the instrument to correct the signals to read each dye in the corresponding channel and to exclude to read signals from neighbored fluorophores. The instrument has to be calibrated with samples containing pure dyes, performing a single dye Real-Time-PCR run and read the fluorescence.
 

Contents

The Color Compensation Set contains 6 vials with premixed lyophilized primers, a Hydrolysis Probe with dark quencher and DNA target for 15 reactions each:

1 Vial green 500 Cyan500
1 Vial yellow 530 Fluorescein (FAM)
1 Vial red 580 Rhodamin 6G YAK, VIC, HEX, Tamra
1 Vial lilac 610 LightCycler Red 610 ROX, Texas
1 Vial blue 640 LightCycler Red 640
1 Vial black 670 LightCycler Red 670


Additional Reagents and Materials Required:

LightCycler® FastStart DNA Master HybProbes
or LightCycler® 480 Probes Master
or LightCycler® Multiplex RNA Virus Master

Note: The use of other reagents has not been tested but is expected to work in the same way.
We recommend to use the mastermix which is intended to be utilized later.

LightCycler® 480 Multiwell Plate 96 (White)
or LightCycler® 480 Multiwell Plate 384 (White)

Note: The use of clear multiwell plates is discouraged.

The emission spectra of the fluorophores used with the Real-Time-PCR Instruments overlap, causing fluorescence signals to be recorded in the adjacent channels when used simultaneously. The Color Compensation (CC) is a tool of the instrument to correct the signals to read each dye in the corresponding channel and to exclude to read signals from neighbored fluorophores. The instrument has to be calibrated with samples containing pure dyes, performing a single dye Real-Time-PCR run and read the fluorescence.

The generated CC file is specific for the respective dyes and mastermix used. For a most accurate CC file, perform CC procedure with the same Enzyme Master utilized in the experiments that shall be compensated.

The CC file might work for similar dyes or other master mixes but this must be verified before using it - simply by running single positive samples or controls for all channels and check that there are no significant signals in the neighboured channels recorded.

CC files generated with Multiplex RNA Virus Master or 480 Probes Master - but not FastStart DNA HybProbes Master - can be used to our experience interchanged - but this has to be verified experimentally.

Description
The CC file is generated during a Real-Time-PCR experiment with hydrolysis (TaqMan) probes collecting data from a melting curve. This requires one reaction per dye and a blank. In order to collect reliable data we recommend to run the reactions in triplicates – the respective procedure is described in this manual.

The manual describes the definition of a new format (Hexaplex) with Quant factors different from the default setting, programming of the instrument with melting curve, and instructions to save the CC file.

The Color Compensation can be included in a run with samples (but requires a distinct subset for the CC).

This kit generate CC files devised for LightMix® Kits; any other use is under customer`s responsibility.

Note to z 480 users: Only 5 different dye can be discriminate by this procedure, disregard the dye Cyan500 tube 500 green.