For life science research only. Not for use in diagnostic procedures. Others LightCycler TaqMan Master LSR LightCycler® TaqMan® Master 3.5.5.1.3.10 04735536001 LC TaqMan Master, 480 rcts. LightCycler TaqMan Master 04038377024356 Reagents, kits 1 kit 480 reactions of 20 μl final volume each false 04535286001 LC TaqMan Master, 96 rcts. LightCycler TaqMan Master 04038377020709 Reagents, kits 1 kit 96 reactions of 20 μl final volume each false How this Product WorksLightCycler® TaqMan® Master is a ready-to-use reaction mix designed for the TaqMan® detection format using the LightCycler® Carousel-Based System.It contains FastStart polymerase for a "Hot Start" PCR, which has been shown to significantly improve the specificity and sensitivity of PCR by minimizing the formation of non-specific amplification products [Chou Q, et al, 1992 and Kellogg DE, et al, 1994]. FastStart Taq DNA Polymerase is a chemically modified form of thermostable recombinant Taq DNA polymerase that is inactive at +15 to +25°C and below. The enzyme is active only at high temperatures, where primers no longer bind non-specifically. The enzyme is completely activated (by removal of blocking groups) in a single pre-incubation step (95°C, 10 minutes) before cycling begins. Activation does not require the extra handling steps typical of other "Hot Start" techniques.The LightCycler® TaqMan® Master provides convenience, excellent performance, reproducibility, and minimal contamination risk. All you have to supply is PCR primers, a detection probe and your template DNA.The reaction mix in this kit is optimized for a fixed MgCl2 concentration, which works with nearly all primer combinations. You do not need to adjust the MgCl2 concentration to amplify different sequences. The performance of the kit described in this Instruction Manual is guaranteed only when it is used with the LightCycler® Carousel-Based System.Test PrincipleHydrolysis probe assays, also called TaqMan® Assays, use a single probe containing two labels, a fluorescent reporter dye and a fluorescent quencher. While the probe is intact, the quencher is close to the reporter dye and suppresses the reporter fluorescence via fluorescence resonance energy transfer (FRET). When the probe is hybridized to the target sequence, the 5'-nuclease activity of the polymerase can cleave the hydrolysis probe, separating the reporter and the quencher. With a rising amount of target sequence during PCR, more probe is cleaved and the fluorescence signal of the unquenched reporter dye increases.As the principle of Hydrolysis probe assays is probe cleavage during PCR, Hydrolysis probes cannot be used to perform a melting curve analysis. In contrast, HybProbe probes which consist of two specially designed, sequence-specific oligonucleotide probes labeled N  with different dyes, are still intact at the end of amplification, and thus may be used in a subsequent melting curve experiment (e.g., for mutation detection or SNP analysis). Two-Step RT-PCRThe LightCycler® TaqMan® Master can also be used to perform two-step RT-PCR.In two-step RT-PCR, the reverse transcription of RNA into cDNA is separated from the other reaction steps and is performed outside the LightCycler® Carousel-Based System. Subsequent amplification and online monitoring is performed according to the standard LightCycler® Carousel-Based System procedure, using cDNA as starting sample material.One of the following reagents is required for reverse transcription of RNA into cDNA:Transcriptor Reverse TranscriptaseTranscriptor First Strand cDNA Synthesis Kit Transcriptor High Fidelity cDNA Synthesis Kit Transcriptor Universal cDNA MasterFirst Strand cDNA Synthesis Kit for RT-PCR (AMV)Synthesis of cDNA is performed according to the detailed instructions provided with the cDNA synthesis reagent.Do not use more than 8 μl of undiluted cDNA template per 20 μl final reaction volume, because greater amounts may inhibit PCR. For initial experiments, Roche recommends running undiluted, 1:10 diluted and 1:100 diluted cDNA templates, in parallel to determine the optimal template amount. en The LightCycler® TaqMan® Master is designed for life science research. In combination with the LightCycler® Carousel-Based System, the kit enables high sensitive detection and quantification of defined DNA sequences (with suitable PCR primers and detection probes). It can also be used to detect and quantify defined RNA sequences in a two-step RT-PCR (with additional reagents for reverse transcription). en Ready-to-use Hot Start Reaction Mix for PCR, using TaqMan® probes with the LightCycler® Carousel-Based System. en