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Vial / Bottle	Cap	Label	Function	Catalog Number	Content
1a	red	LightCycler® FastStart DNA Master HybProbe, LC FastStart Enzyme	Ready-to-use hot start reaction mix after pipetting 60 μl from Vial 1b into one Vial 1a Contains FastStart Taq DNA Polymerase, reaction buffer, dNTP mix with dUTP instead of dTTP, and 10mM MgCl2.	03 003 248 001	3 vials 1a and 3 vials 1b, for 3 × 64 μl each LightCycler® FastStart DNA Master HybProbe, 10x conc.
1b	colorless	LightCycler® FastStart DNA Master HybProbe, LC FastStart Reaction Mix HybProbe, 10x conc.
12 239 272 001	15 vials 1a and 15 vials 1b, for 15 × 64 μl each LightCycler® FastStart DNA Master HybProbe, 10x conc.
2	blue	LightCycler® FastStart DNA Master HybProbe, MgCl2 stock solution, 25 mM	To adjust MgCl2 concentration in the reaction mix.	03 003 248 001	1 vial, 1 ml
12 239 272 001	2 vials, 1 ml each
3	colorless	LightCycler® FastStart DNA Master HybProbe, Water, PCR Grade	To adjust the final reaction volume.	03 003 248 001	2 vials, 1 ml each
12 239 272 001	7 vials, 1 ml each

", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "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:

1. The donor dye probe has a fluorescein label at its 3' end and the acceptor dye probe has a red fluorophore label [LightCycler^® Red 610¹, LightCycler^® Red 640, Cy5 {670}¹, or Cy5.5 {705}] at its 5' end it is 3'-phosphorylated, so it can not 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.
    
2. 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.
    
3. After annealing, an increase in temperature leads to elongation and displacement of the probes.
    
4. 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.

1. LightCycler^® Red 610 and Cy5 {670} can only be used on a LightCycler^® 2.0 Instrument.

This kit is ideally suited for hot start PCR applications in glass capillaries. In combination with the LightCycler^® Carousel-Based System and 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^® Carousel-Based 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.


Hot start PCR has been shown to significantly improve the specificity and sensitivity of PCR by minimizing the formation of non-specific amplification products at the beginning of the reaction (Chou Q. et al., 1992; Kellogg, D.E. et al., 1994; Birch, D.E. et al., 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, 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.
 ", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "

Procedure	Assay Time [min]
Optional: Dilution of template DNA	5
PCR Set-up	15
LightCycler®Carousel-Based System run incl. Melting Curve	45
Total assay time	65

", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "LightCycler^® FastStart DNA Master HybProbe is an easy-to-use hot start reaction mix for sensitive PCR applications in LightCycler^® Capillaries, 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.", "Country": "XG", "Code": "Applications", "Name": "Applications" } ] } } ] }