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"Storage Conditions (Product)", "Name": "Storage Conditions (Product)" }, { "Language": "en", "Value": "+2 to +8°C", "Country": "XG", "Code": "Storage Conditions (Working Solution)", "Name": "Storage Conditions (Working Solution)" }, { "Language": "en", "Value": "HybProbe probes

HybProbe probes are two sequence-specific hybridization probes labeled with fluorescent dyes that are designed to bind close together on a single DNA strand. The detection principle of dual hybridization probes is called fluorescence resonance energy transfer (FRET). FRET involves the transfer of energy from a donor fluorophore on one probe to an acceptor fluorophore on the other. If the donor and the acceptor fluorophore are very close together, excitation of the donor by the light source of the PCR instrument results in energy transfer to the acceptor, which emits light of a longer wavelength that can be measured.

HybProbe probes are designed as a pair with one probe labeled with the donor dye (fluorescein) and the other probe labeled with the acceptor dye (LightCycler® Red 610, LightCycler® Red 640, Cy5 or Cy5.5). For dual-color reactions, typically LightCycler® Red 640 and Cy5.5 are used as the acceptor dyes.

In a typical amplification reaction, the reaction mix is first heated to separate (denature) the double stranded template DNA. The temperature is then lowered for annealing of the primers. At this temperature, HybProbe probes bind close together on complementary sequences of one of the single DNA strands. This binding, or hybridization, of a dye labeled probe pair results in energy transfer between the fluorescent dyes of the two probes. The real-time PCR instrument measures the fluorescence signal in each PCR cycle. Increasing amounts of fluorescence during an amplification reaction indicate increasing amounts of DNA.


Color compensation

The LightCycler® Carousel-Based System simultaneously detects and analyzes more than one color in each capillary. Color compensation is necessary when dual and/or multicolor assays are run in a single capillary. Fluorescent dyes emit over a broad range of wavelengths, and there is some overlap of their emission spectra, also known as signal crosstalk. Color compensation corrects for this spectral overlap by subtracting crosstalk of fluorescent signals outside the dominant channel.

The following factors can influence the degree of fluorescent signal crosstalk:
  • Temperature
  • Coupling chemistry of the fluorescent dyes
  • Properties of the optical system

To compensate for temperature, a color compensation calibration run includes a temperature gradient step (+40 to +95°C), in which data from all fluorescent dyes are continuously measured in all channels.
To minimize the effects of coupling chemistry, ensure that labeled probes are of high quality. Optical systems show minor variations from instrument to instrument. To compensate for the properties of the optical system, generate and store instrument-specific color compensation data for the fluorophores that will be used.
Use the LightCycler® Color Compensation Set to generate color compensation data. This is a prerequisite for performing dual-color experiments with both LightCycler® Red 640-labeled and Cy5.5-labeled HybProbe probes in a single capillary. The generated color compensation file/key can universally be used in all subsequent dual-color experiments on the specific LightCycler® Instrument, to compensate for the raw fluorescence data either online during the run, or for data analysis after the run.", "Country": "XG", "Code": "Background Information - Help Corner", "Name": "Background Information - Help Corner" }, { "Language": "en", "Value": "The LightCycler® Control Kit DNA allows performance of a control reaction for PCR using the LightCycler® Carousel-Based System. A 110 bp fragment of the β-globin gene is amplified with specific primers from human genomic DNA. Amplification and online monitoring of the control gene is achieved by a combined procedure on the LightCycler® System. The results are interpreted directly after completing the PCR. The amplicon is detected by fluorescence using either the dsDNA intercalating dye SYBR Green I or a β-globin-specific pair of HybProbe probes provided in the kit.
​​​​​​​
How this Product Works
The LightCycler® Control Kit DNA allows control reaction for PCR to be performed using the LightCycler® System and the respective LightCycler® Kit (LightCycler® DNA Master, LightCycler® DNA FastStart Master, or LightCycler® DNA FastStart MasterPLUS).
For detection using SYBR Green I, a 110 bp fragment of the human β-globin gene is amplified with specific primers in the presence of this DNA double-strand specific dye. During online monitoring, the fluorescence increases as a result of the increasing amount of PCR product. Identification of the correct PCR product is done by Melting Curve Analysis.
For detection using HybProbe probes, a 110 bp fragment of the human β-globin gene is amplified with specific primers from human genomic DNA. Amplification of target DNA is monitored using HybProbe pairs that hybridize to an internal sequence of the amplified fragment. HybProbe probes are two different short oligonucleotides that bind to an internal sequence of the amplified fragment during the annealing phase of the amplification cycle. One probe is labeled at the 5´-end with a LightCycler® Red fluorophore LightCycler® Red 640 and Cy5.5; it is also 3´-phosphorylated, so it cannot be extended. The other probe is labeled at the 3´-end with fluorescein. When hybridized to the template DNA, the two probes are close enough to allow fluorescence resonance energy transfer FRET between the two fluorophores.
During FRET, fluorescein the donor fluorophore is excited by the light source of the LightCycler® Instrument. Fluorescein transfers part of this excitation energy to the LightCycler® Red dye the acceptor fluorophore. Then, the LightCycler® Red dye emits fluorescence, which is measured by the LightCycler® Instrument. HybProbe probes that contain different LightCycler® Red labels can be used separately for single color detection experiments or combined for dual color detection experiments. Color compensation is not necessary for single color detection experiments. However, if you are using HybProbe probes to perform dual color experiments in a single capillary, you must also use a color compensation file. Color compensation may be applied either during or after a run on the LightCycler® Instrument.", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "Always run a negative control with the samples.
To prepare a negative control, replace the template DNA with Water, PCR Grade (Vial 5).", "Country": "XG", "Code": "Control Reactions", "Name": "Control Reactions" }, { "Language": "en", "Value": "
ProcedureAssay Time [min]
Preparing a fresh dilution series of the human genomic DNA5
PCR Set-up15
LightCycler®Carousel-Based System run
depending on protocol for the dedicated reagent
45
Total assay time65
", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "The LightCycler® Control Kit DNA is designed for use in research studies and includes human genomic DNA as the control template, control primers, HybProbe mixes, and Water, PCR Grade, for performing PCR using the LightCycler® Instruments. It is designed as a control to confirm PCR system performance using the LightCycler® System or as a quality check of the human DNA used in a specific assay, when combined with the function-tested primers and HybProbe mixes provided with the kit.
Alternatively, the kit can be used as a quality check of specific primers and HybProbe probes by using the human genomic DNA provided with the kit. Furthermore, the kit can be used as a functional check of dual-color experiments and color compensation when simultaneously using the provided LightCycler®  Red 640- and Cy5.5-labeled HybProbe pairs.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "Kit for performing PCR control reactions using the LightCycler® Carousel-Based System.", "Country": "XG", "Code": "Product Characteristics", "Name": "Product Characteristics" }, { "Language": "en", "Value": "The LightCycler® Control Kit DNA includes human genomic DNA as the control template, control primers, HybProbe probes and PCR-grade water for performing PCR using the LightCycler® Carousel-Based System. It is designed to confirm PCR performance using LightCycler® Carousel-Based Instruments.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "
VialCapLabelFunction / DescriptionContent
1purpleLightCycler® Control Kit DNA, Human Genomic DNATemplate for control reaction [15 ng/μl]3 vials, 50 μl each
2whiteLightCycler® Control Kit DNA, β-Globin Primer Mix, 10x conc.
  • Ready-to-use primer mix specific for the human β-globin gene [5 μM]
  • Unlabeled, 20-mer primers
  • For amplification of the human β-globin gene using either SYBR Green I or HybProbe detection format
2 vials,  50 μl each 

3yellowLightCycler® Control Kit DNA, β-Globin HybProbe Mix,
LightCycler® Red 640-labeled, 10x conc.
  • Ready-to-use HybProbe mix, specific for human β-globin gene
  • Probe 1: 35-mer, fluorescein-labeled at the 3'-end, [2 μM]
  • Probe 2: 29-mer, LightCycler® Red 640-labeled at the 5'-end and phosphorylated at the 3'-end, [4 μM]
  • For detection of the human β-globin control PCR product
2 vials,  50 μl each
4yellowLightCycler® Control Kit DNA, β-Globin HybProbe Mix,
Cy5.5-labeled, 10x conc.
  • Ready-to-use HybProbe mix, specific for human β-globin gene
  • Probe 1: 35-mer, fluorescein-labeled at the 3'-end, [2 μM]
  • Probe 2: 29-mer,  Cy5.5-labeled at the 5'-end and phosphorylated at the 3'-end, [4 μM]
  • For detection of the human β-globin control PCR product
2 vials,  50 μl each
5colorlessLightCycler® Control Kit DNA, Water, PCR GradeFor adjusting reaction volume2 vials,  1 ml each
", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "
Standard Laboratory Equipment
  • Nuclease-free, aerosol-resistant pipette tips
  • Sterile reaction tubes for preparing master mixes and dilutions
For PCR
  • LightCycler® Systems* 
  • LightCycler® Capillaries*
  • Standard benchtop microcentrifuge containing a rotor for 2.0 ml reaction tubes
The LightCycler® System provides adapters that allow LightCycler® Capillaries to be centrifuged in a standard microcentifuge rotor.
or
  • LC Carousel Centrifuge 2.0* for use with the LightCycler® 2.0 Carousel optional
If you use a LightCycler® Instrument version below 2.0, you need in addition the LC Carousel Centrifuge 2.0 Bucket 2.1*. To adapt the Light-Cycler® 2.0 Carousel to the former LC Carousel Centrifuge, you need the LC Carousel Centrifuge 2.0 Rotor Set*.

  • LightCycler® Uracil-DNA Glycosylase* or Uracil-DNA Glycosylase, heat-labile* (optional)
For prevention of carryover contamination; see section Prevention of carryover contamination. Use LightCycler® Uracil-DNA Glycosylase in combination with LightCycler® FastStart Masters only.

​​​​​​​Reagents for PCR using SYBR Green I detection format
  • LightCycler® FastStart DNA Master SYBR Green I
  • LightCycler® FastStart DNA MasterPLUS SYBR Green I
Reagents for PCR using HybProbe detection format
  • LightCycler® DNA Master HybProbe
  • LightCycler® FastStart DNA Master HybProbe
  • LightCycler® FastStart DNA MasterPLUS HybProbe
  • LightCycler® Color Compensation Set* optional
 
To perform color compensation when using the LightCycler® Red 640- and Cy5.5-labeled HybProbe probes in dual-color experiments in the same capillary, see section Color Compensation.
", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" } ] } } ] }

LightCycler® Control Kit DNA

Kit for performing PCR control reactions using the LightCycler® Carousel-Based System.

LightCycler<sup><sup>®</sup></sup> Control Kit DNA

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