LightCycler^® 480 System Reagents

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General Concept
Benefits
Easy-to-Use One Component Master Mixes
Hot Start Enzymes for Optimized Specificity and Sensitivity
Carryover Prevention
Developed for or Readily Combined with RT-PCR
Compatibility

The LightCycler^® 480 Real-Time PCR System includes generic master mixes tailor-made for each key application in genomics research (gene identification, gene quantification, variation screening, and genotyping).

Enzyme variants and buffer conditions have been carefully chosen and optimized for each application:

• LightCycler^® 480 SYBR Green I Master contains components which help minimize primer-dimers.
• LightCycler^® 480 Genotyping Master is optimized for melting curve analysis with HybProbe or SimpleProbe probes, especially in multiplex assays.
• LightCycler^® 480 High Resolution Melting Master, based on the saturating DNA dye ResoLight, allows heteroduplex analysis for mutation scanning and genotyping. LightCycler^® 480 RNA Master Hydrolysis Probes enables ultra-fast, sensitive one-step RT-PCR.

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• True, user-friendly one-component master mixes for exceptional sensitivity in fast PCR applications.
• Optimized shapes (slopes, heights) for amplification and melting curve, allowing bias-free Cp- and T_m-determination, respectively.
• Combination with highly reproducible LightCycler^® 480 thermal block cycler and optimized disposables eliminates the need for passive reference dyes.
• LightCycler^® 480 SYBR Green I Master and Probes Master available in pack sizes suitable for high-throughput applications.
• Extended stability at room temperature (see Figure 1) and at +4° to +8°C for up to four weeks, for added convenience with daily or routine use (LightCycler^® 480 SYBR Green I and LightCycler^® 480 Probes Master).

Figure 1: Stability of the LightCycler^® 480 SYBR Green I Master. PCR reactions of serial 1:10 dilutions (10,000–10 copies/reaction) with three replicates of a human DNA target sequence were set up. The amplification reactions were then run either immediately or after 24 hours incubation on a loading robot at room temperature. The amplification curves and the corresponding Cps of the directly-assayed (blue-colored curves) and delayed-assayed (red-colored curves) samples demonstrate that the performance of the hot start enzyme master mix was not affected by prolonged standing.

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Since all mixes are provided as one-component master reagents, reaction setup requires only the addition of template DNA, primers, and except for experiments with SYBR Green I, probes.

The mixes can be used with different types of DNA (e.g., genomic, cDNA) and are ideally suited for high-throughput applications in 96- or 384-well plates.

Each master mix is optimized for a fixed MgCl₂ concentration, which works with nearly all primer combinations. No adjustment of the MgCl₂ concentration is needed to amplify different sequences.

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LightCycler^® 480 PCR master mixes are all based on enzyme compatible with hot start protocols. When used on the LightCycler^® 480 Instrument, these protocols have been shown to significantly improve the specificity, sensitivity, and yield of PCR. For example, heat-labile blocking groups on some of the amino acid residues of FastStart Taq DNA Polymerase make the modified enzyme inactive at room temperature. Therefore, there is no elongation during the period when primers can nonspecifically bind. The FastStart Taq DNA Polymerase is activated by removing the blocking groups at a high temperature (i.e., a pre-incubation step at +95°C).

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LightCycler^® 480 PCR master mixes contain a dNTP mix including UTP instead of dTTP. Therefore, they can be used in conjunction with heat-labile Uracil DNA Glycosylase for carryover prevention during PCR.

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LightCycler^® 480 master mixes can be used in two-step RT-PCR applications, for example, downstream of Transcriptor Reverse Transcriptase.

For one-step reactions, the LightCycler^® 480 RNA Master Hydrolysis Probes is available.

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The LightCycler^® 480 master mixes have been adapted to the special rapid cycling environment of the LightCycler^® 480 Instrument and to the different probe chemistries supported by the system. Optimal experimental results can therefore only be obtained when the LightCycler^® 480 Instrument and reagents are used in combination. Due to the differences in design (dimensions, material) of the respective reaction devices, reagents are tailor-made and must not be interchanged between the LightCycler^® 480 and the classical, capillary-based LightCycler^® 2.0 and 1.5, or the new strip-based LightCycler^® Nano System.

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