For general laboratory use. Others LightCycler FastStart DNA Master HybProbe GLU LightCycler® FastStart DNA Master HybProbe 3.5.5.1.3.2 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. The basic steps of DNA detection by HybProbe probes during real-time PCR on the LightCycler® System are: The donor dye probe has a fluorescein label at its 3' end and the acceptor dye probe has a red fluorophore label (Red 610, Red 640, Cy5, or Cy5.5) at its 5' end it is 3'-phosphorylated, so it cannot be extended. Hybridization does not occur during the Denaturation phase of PCR. As the distance between the unbound dyes prevents energy transfer, no fluorescence will be detected from the red acceptor dye during this phase. During the Annealing phase, the probes hybridize to the amplified DNA fragment in a head-to-tail arrangement, thereby bringing the two fluorescent dyes close to each other. Fluorescein is excited by the light source of the LightCycler® System, which causes it to emit green fluorescent light. The emitted energy excites the red fluorophore acceptor dye by fluorescence resonance energy transfer FRET. The red fluorescence emitted by the acceptor dye is measured at the end of each annealing step, when the fluorescence intensity is greatest. After annealing, an increase in temperature leads to elongation and displacement of the probes. At the end of the Elongation step, the PCR product is double-stranded, while the displaced HybProbe probes are back in solution and too far apart for FRET to occur. HybProbe probes that carry different red fluorophore labels can be used separately for single-color detection experiments, or combined for dual- or multiple-color detection experiments. The LightCycler® PRO Instrument does not require the creation of a color compensation key. For the LightCycler® Carousel-Based System, color compensation is not necessary for single-color detection experiments. However, if using HybProbe probes to perform dual- or multiple-color experiments in a single capillary, a color compensation file must be used. Color compensation may be applied either during or after a run on the LightCycler® Carousel-Based System. See the LightCycler® Instrument Operator’s Manual and the Instructions for Use of the LightCycler® Color Compensation Set for more information on the generation and use of a color compensation file or key. How this product works This kit is ideally suited for hot start PCR applications. Using suitable PCR primers and HybProbe probes, this kit enables very sensitive detection and quantification of defined DNA sequences. It can also be used to genotype single nucleotide polymorphisms SNPs and analyze mutations using Melting Curve analysis. Furthermore, this kit can be used to perform two-step RT-PCR, in combination with a reverse transcription kit for cDNA synthesis*. In principle, LightCycler® FastStart DNA Master HybProbe can be used for the amplification and detection of any DNA or cDNA target. However, the amplification protocol must be optimized to the reaction conditions of the LightCycler® System and specific PCR primers and HybProbe probes designed for each target. LightCycler® FastStart DNA Master HybProbe can also be used with LightCycler® Uracil-DNA Glycosylase, to prevent carryover contamination during PCR. The amplicon size should not exceed 750 bp in length. For optimal results, select a product length of 500 bp or less. Hot start PCR has been shown to significantly improve the specificity and sensitivity of PCR by minimizing the formation of nonspecific amplification products at the beginning of the reaction. FastStart Taq DNA Polymerase is a chemically modified form of thermostable recombinant Taq DNA polymerase, that shows no activity up to +75°C. 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. LightCycler® FastStart DNA Master HybProbe provides convenience, excellent performance and reproducibility, as well as minimal contamination risk. All that is required is template DNA, PCR primers, HybProbe probes and additional MgCl2 if necessary. en LightCycler® FastStart DNA Master HybProbe is an easy-to-use hot start reaction mix for sensitive PCR applications using HybProbe probes as detection format. It is an ideal master mix for performing quantitative PCR as well as SNP and mutation detection, and can also be used in two-step RT-PCR.LightCycler® FastStart DNA Master HybProbe can also be used with LightCycler® Uracil-DNA Glycosylase to prevent carryover contamination during PCR. en

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LightCycler^® FastStart DNA Master HybProbe

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