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Procedure	Assay Time [min]
PCR Setup	15
Reverse Transcription	10
LightCycler®Carousel-Based System PCR run	25
Total Assay Time	50

", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "For designing HybProbe probes, please refer to LightCycler^® Technical Note 6/99 “Selection of Hybridization Probe Sequences for Use with the LightCycler”. In addition, LightCycler^® Probe Design Software 2.0 can identify the best HybProbe probe and primer combinations and provide precise estimations of the T_ms of the primers and probes in LightCycler^® Reagent Kits.


Recommended HybProbe Probes for the LightCycler^® Carousel-Based System

The LightCycler^® 1.5 Instrument has 3 channels: F1 [530], F2 [640] and F3 [705], for dual-color experiments using HybProbe probes labeled with LightCycler^® Red 640 [640] and Cy5.5 [705].

The LightCycler^® 2.0 Instrument has 6 channels: 530, 560, 610, 640, 670 and 705, for multi-color experiments using HybProbe probes labeled with LightCycler^® Red 610, LightCycler^® Red 640, Cy5 {670} and Cy5.5 {705}.



Difference between HybProbe probes and Hydrolysis/TaqMan^® probes

HybProbe probes are not cleaved like hydrolysis probes, instead they are displaced. The ‘normal’ reaction of Taq DNA Polymerase during synthesis is to displace. The polymerase will cleave a probe only if it has certain characteristics and if the annealing/elongation temperature is +60°C, which is the temperature that the 5’-3’ exonuclease activity of the polymerase is optimal.
Please Note: In contrast to HybProbe probes, hydrolysis probes are cleaved during PCR and thus, can not be used to perform a Melting Curve analysis.


Performing a Melting Curve analysis with HybProbe probes can identify and characterize PCR products using their specific melting behavior for genotyping and mutation analysis.



The principle of Melting Curve analysis is:

1. At the beginning of a melting curve analysis, the reaction temperature is low and the fluorescence signal is high.

2. As the temperature steadily increases, the fluorescence will suddenly drop as the reaction reaches the melting temperature (T_m) of each DNA fragment.

3. A graph of melting behavior [plot of the first negative derivative (-dF/dT) of the fluorescence vs. temperature] reveals that pure, homogeneous PCR products produce a single, sharply defined melting curve with a narrow peak. In contrast, primer-dimers melt at relatively low temperatures and have broader peaks.



Crossing Point (Cp)

1. In an amplification reaction, the cycle at which the fluorescence of a sample rises above the background fluorescence is called the \"crossing point\" (Cp) of the sample.

2. The Cp of a sample appears as a sharp upward curve on the display of the experiment's fluorescence chart.

3. The Cp is the point (PCR cycle number) at which the amplified product is first visible.

4. A sample's Cp depends on the initial concentration of DNA in the sample. A sample with a lower initial concentration of target DNA requires more amplification cycles to reach the Cp. A sample with a higher concentration requires fewer cycles to produce the Cp.


The software uses the calculated Cps of the standard samples to generate the standard curve of Cp versus sample concentration.

", "Country": "XG", "Code": "Background Information - Help Corner", "Name": "Background Information - Help Corner" }, { "Language": "en", "Value": "

Vial/Bottle	Cap	Label	Function / Description	Content
1	red	LightCycler® RNA Amplification Kit HybProbe, LC RT-PCR Enzyme Mix	Enzyme mix for RT-PCR.	2 vials, 20 μl each
2	red	LightCycler® RNA Amplification Kit HybProbe, LC RT-PCR Reaction Mix, 5x conc.	Reaction mix for RT-PCR. Contains reaction buffer, dNTP mix (with dUTP instead of dTTP), and 15 mM MgCl2.	3 vials, 128 μl each
3	blue	LightCycler® RNA Amplification Kit HybProbe, MgCl2 stock solution, 25 mM	To adjust MgCl2 concentration.	1 vial, 1 ml
4	colorless	LightCycler® RNA Amplification Kit HybProbe, Water, PCR Grade	To adjust the final reaction volume.	2 vials, 1 ml each

", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "The LightCycler^® RNA Amplification Kit HybProbe is designed specifically for the HybProbe detection format using the LightCycler^® Carousel-Based System. The kit provides reagents, including RT-PCR enzyme mix, reaction mix, MgCl₂, and Water, PCR Grade for very sensitive detection and quantification of defined RNA sequences using the LightCycler^® System (suitable RT-PCR primers and HybProbe probes must be supplied). It can also be used to genotype single nucleotide polymorphisms (SNPs) and analyze mutations.

In addition, the kit can be used with Uracil-DNA Glycosylase (UNG), heat labile to prevent carryover contamination during PCR.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "

• Nuclease-free, aerosol-resistant pipette tips

• Sterile reaction tubes for preparing master mixes and dilutions

• LightCycler^® Carousel-Based System*
• LightCycler^® Capillaries*
• Standard benchtop microcentrifuge, containing a rotor for 2.0 ml reaction tubes

or

• LC Carousel Centrifuge 2.0* for use with the LightCycler^® 2.0 Sample Carousel (20 μl; optional)
• Uracil-DNA Glycosylase, heat-labile* (optional)

• LightCycler^® Color Compensation Set* (optional)

", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" }, { "Language": "en", "Value": "Absolute fluorescence values in samples containing the same starting amount of template can vary. The most common reason is a difference in pipetting between samples. It is important to remember that absolute fluorescence readings for a sample are arbitrary and do not affect analysis of Cp (crossing point) determination or melting curve analysis in later data analysis.

During the seek process, the first capillary position is always found by the instrument, however, the capillaries thereafter must exceed a certain fluorescence background level (the \"seek threshold\") to be recognized by the instrument. The main reason why capillaries are not found during the initial seek process is that the fluorescent dye has either been omitted or degraded. To prevent degradation of the HybProbe probes, keep them away from light!

Performing a 4-step Melting Curve can help when both probes have very different melting temperatures, to ensure that the probes bind correctly and that enough probe is bound for detection.
An example of a 4-step Melting Curve may be as follows:
+95°C for 0 sec
+59°C for 15 sec
+45°C for 15 sec
+95°C for 0 sec with a Ramp Rate of 0.1°C/sec and continuous acquisition.", "Country": "XG", "Code": "Troubleshooting - Help Corner", "Name": "Troubleshooting - Help Corner" }, { "Language": "en", "Value": "Easy-to-use Reaction Mix for One-Step RT-PCR, using HybProbe probes with the LightCycler^® Carousel-Based System.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "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 fluorophore dye (LightCycler^® Red 610, 640, Cy5, or 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 dye (the acceptor fluorophore). Then, the dye emits fluorescence which is measured by the LightCycler^® Instrument. HybProbe probes  that contain different dye 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.The LightCycler^® RNA Amplification Kit HybProbe is designed specifically for the HybProbe detection format using the LightCycler^® Carousel-Based System. It is used to perform one-step RT-PCR in 20 μl glass capillaries. Amplification and online monitoring of the template RNA is achieved by a combined procedure on the LightCycler^® Instruments. The results are interpreted directly after completing the PCR. The amplicon is detected by fluorescence using target-specific HybProbe probes (not supplied with the kit).
The LightCycler^® RNA Amplification Kit HybProbe provides convenience, high performance, reproducibility, and minimizes contamination risk. Only template RNA, PCR primers, HybProbe probes, and additional MgCl₂ (if necessary), need to be added.

In principle, the LightCycler^® RNA Amplification Kit HybProbe can be used for the amplification and detection of every RNA target. However, you would need to adapt each amplification protocol to the reaction conditions of the LightCycler^® Carousel-Based System and design specific PCR Primers and HybProbe probes for each target. See the LightCycler^® Operator's Manual for general recommendations.
​​​​​​​", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "During cycling, Fluorescence readings for HybProbe probes should be taken at the end of annealing. In the LightCycler^® Software, the Acquisition Mode should be ‘Single’ in the Annealing step.

For Melting Curve, the Acquisition Mode can be ‘Continuous’ or ‘Step’.
In the ‘Continuous’ mode, fluorescence data is acquired continuously as the temperature changes, with the rate of data acquisition dependent on the number of capillaries.
In the ‘Step’ mode, fluorescence data is acquired at each temperature transition. For example, if the Ramp Rate is 0.5°C/sec, fluorescence data is acquired every 0.5°C for every capillary.

Channel Settings
To correct for pipetting errors in assays using HybProbe probes, as the variation will occur in both channels, a division by the non-reporting signal from Channel 530 will compensate for potential difference between samples and standards.

• The 530 option uses the values of every single data point in Channel 530 to correct the respective data point in the red fluorophore channel.
• The Back 530 option refers to the signal from Channel 530 in cycles 2 to 6, thus this option is used for dual- or multi-color experiments. Due to the presence of several Fluorescein labeled probes in dual- and multi-color experiments, the corresponding signals from channel 530 can not be used to correct the signals of the reporter channels.

For amplification analysis of HybProbe probes, the channel setting should be divided by ‘530’ for mono-color experiments and by ‘Back 530’ for dual- or multi-color experiments.

For Melting Curve analysis, it is not necessary to divide by ‘530’ or ‘Back 530’.

For further information regarding Channel Settings, please refer to the LightCycler^® Carousel-Based System LightCycler^® Software 4.1 Addendum 1 - February 2010.", "Country": "XG", "Code": "Protocols - Help Corner", "Name": "Protocols - Help Corner" } ] } } ] }