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These probes hybridize to their complementary sequence in target PCR products. Hydrolysis probe chemistry uses the so-called FRET principle. Fluorescence Resonance Energy Transfer (FRET) is based on the transfer of energy from one fluorophore (the donor or reporter) to another adjacent fluorophore (the acceptor or quencher).
Hydrolysis probe assays can technically be described as homogeneous 5'-nuclease assays, since a single 3' non-extendable probe, which is cleaved during PCR amplification, is used to detect the accumulation of a specific target DNA sequence (Holland, PM. et al., 1991). This single probe contains two labels, a fluorescent reporter and a quencher, in close proximity to each other. When the probe is intact, the quencher dye is close enough to the reporter dye to suppress the reporter fluorescent signal (fluorescent quenching takes place via FRET). During PCR, the 5'-nuclease activity of the polymerase cleaves the hydrolysis probe, separating the reporter and quencher. The reporter dye is no longer quenched and emits a fluorescent signal when excited.
The LightCycler^® 96 Instrument is factory calibrated for the following commonly used reporter dyes for hydrolysis probes: FAM, VIC, HEX, Yellow 555, LightCycler^® Red 610, Texas Red, and Cy5. These labeled hydrolysis probes can be used separately or in combination, which permits up to four-color (LightCycler^® 96 Instrument) detection. There is no need for color compensation/calibration runs.FastStart Essential DNA Probes Master is a ready-to-use reaction mix specifically developed for the hydrolysis probe detection format in LightCycler^® 480 Multiwell Plates 96, or 8-Tube Strips on the LightCycler^® 96 Instrument. It contains FastStart Taq DNA Polymerase for hot start PCR, which significantly improves the specificity and sensitivity of PCR by minimizing the formation of nonspecific amplification products (Chou, Q., et al., 1992, Kellogg, D.E., et al., 1994, Birch, D.E., 1996).
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, 5 to 10 minutes) before cycling begins. Activation does not require the extra handling steps typical of other hot start techniques.", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "FastStart Essential DNA Probes Master is a ready-to-use reaction mix containing FastStart Taq DNA Polymerase for hot start PCR, which significantly improves the specificity and sensitivity of PCR by minimizing the formation of nonspecific amplification products. The 2x master mix is optimized for a fixed MgCl₂ concentration, which works with nearly all primer combinations.
No adjustment in the MgCl₂ concentration is needed to amplify different sequences; only template DNA, PCR primers, and hydrolysis probes (e.g., from the Universal ProbeLibrary) must be added.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "Ready-to-use hot start reaction mix for real-time PCR with the LightCycler^® 96 System.", "Country": "XG", "Code": "Product Characteristics", "Name": "Product Characteristics" }, { "Language": "en", "Value": "The FastStart Essential DNA Probes Master is designed for real-time PCR using the LightCycler^® 96 and Nano Instruments in combination with suitable probes (e.g., hydrolysis probes, Universal ProbeLibrary probes, and others) and gene-specific primers. The kit is ideally suited for hot start PCR assays for gene quantification.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "

Vial / Bottle	Cap	Label	Function / Description	Catalog Number	Content
1	red	FastStart Essential DNA Probes Master, 2x conc.	Ready-to-use hot start PCR mix. Contains FastStart Taq DNA polymerase, reaction buffer, dNTP mix (with dUTP instead of dTTP), and MgCl2.	06 402 682 001	5 vials, 1 ml each
06 924 492 001	10 vials, 5 ml each
2	colorless	FastStart Essential DNA Probes Master, Water, PCR Grade	To adjust the final reaction volume.	06 402 682 001	5 vials, 1 ml each
06 924 492 001	2 vials, 25 ml each

", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "Variable, depending on the number of cycles and the annealing time. For example, if the cycling program specifies 45 cycles with 10 seconds denaturation and 30 seconds annealing, a LightCycler^® 96 PCR run will last about 1:12 hours, including 10 minutes pre-incubation time.", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" } ] } } ] }