For general laboratory use. Others LightCycler DNA Master HybProbe GLU LightCycler® DNA Master HybProbe 3.5.5.1.1.2 12158825001 LC DNA Master Hy.Pb, 480 react. LightCycler DNA Master HybProbe 04038377016689 Reagents, kits 1 kit 480 reactions of 20 μl final volume each false 12015102001 LC DNA Master Hy. Pb, 96 react. LightCycler DNA Master HybProbe 04038377016436 Reagents, kits 1 kit 96 reactions of 20 μl final volume each false Easy-to-use reaction mix for PCR, using HybProbe probes with the LightCycler® Carousel-Based System. en LightCycler® DNA Master HybProbe is suited for PCR applications in glass capillaries and with 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.LightCycler® DNA Master HybProbe can also be used with Uracil-DNA Glycosylase, heat-labile, to prevent carryover contamination during PCR. en 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® Carousel-Based System are:The donor dye probe has a fluorescein label at its 3' end and the acceptor dye probe has a red fluorophore label (LightCycler® Red 610(1), LightCycler® Red 640, Cy5 [670](1), or Cy5.5 [705]) 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® Carousel-Based 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). 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.LightCycler® Red 610 and Cy5 [670] can only be used on a LightCycler® 2.0 Instrument.How this Product WorksLightCycler® DNA Master HybProbe is a ready-to-use PCR reaction mix designed specifically for real-time PCR assays using the detection format of the HybProbe probe on the LightCycler® Carousel-Based System. It is used to perform PCR in 20 μl capillaries. LightCycler® DNA Master HybProbe provides convenience, excellent performance and reproducibility, as well as minimal contamination risk. All that is required are template DNA, PCR primers, HybProbe probes, and additional MgCl2 (if necessary).In principle, LightCycler® 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® Carousel-Based System and specific PCR primers and HybProbe probes designed for each target.The amplicon size should not exceed 750 bp in length. For optimal results, select a product length of 500 bp or less. en