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The kit can be used to perform quantitative PCR, as well as SNP and mutation detection. The addition of MgCl₂ (provided in the kit) for specific applications may be necessary.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "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. With HybProbe probes, 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": "Hot Start

If the reaction components are not thoroughly mixed prior to the initial heat denaturation step, non-specific annealing and primer elongation may occur. Conventional manual hot start or wax techniques can not be used with the glass capillaries of the LightCycler^® Carousel-Based System. For hot start PCR, Roche recommends using LightCycler^® FastStart DNA Master HybProbe, or LightCycler^® FastStart DNA Master^PLUS HybProbe, which contain a chemically modified Taq DNA Polymerase, that is activated by heat.

For further information, consult the Roche Applied Science catalog or homepage: www.roche-applied-science.com.



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 of HybProbe probes, 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.



To perform Touchdown PCR with LightCycler^® DNA Master HybProbe, decrease the annealing temperature during the first 20 cycles by 10°C in steps of 2°C: start from an initial temperature +6 to +8°C above the melting temperature of your PCR primers down to a temperature 2°C above the final annealing temperature. Use a Ramp Rate of 0.5°C per cycle. The final 25 cycles are then run at the final annealing temperature.

An example touchdown protocol for a PCR primer set of T_m +56°C might be as follows:
+95°C, 10 min
+95°C, 10 s / +62°C 5 s / +72°C 10 s (x 4 cycles)
+95°C, 10 s / +60°C 5 s / +72°C 10 s (x 4 cycles)
+95°C, 10 s / +58°C 5 s / +72°C 10 s (x 4 cycles)
+95°C, 10 s / +56°C 5 s / +72°C 10 s (x 4 cycles)
+95°C, 10 s / +54°C 5 s / +72°C 10 s (x 4 cycles)
+95°C, 10 s / +52°C 5 s /+ 72°C 10 s (x 25 cycles)

Note that elongation time depends on product length, follow the general rule: elongation time (sec) = amplicon length [bp]/25.



Cloning of PCR products amplified with the LightCycler^® DNA Master HybProbe may be done by direct T/A cloning, blunt-end cloning, or after restriction enzyme digestion. It is not necessary to purify the PCR products prior to cloning. As host, use a UNG-negative E. coli strain, as the LightCycler^® Reagent Kits all contain dUTP.", "Country": "XG", "Code": "Protocols - Help Corner", "Name": "Protocols - Help Corner" }, { "Language": "en", "Value": "Easy-to-use reaction mix for PCR, using HybProbe probes with the LightCycler^® Carousel-Based System.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "

Vial / Bottle	Cap	Label	Function / Description	Catalog Number	Content
1	red	LightCycler® DNA Master HybProbe, LC DNA Master HybProbe, 10x conc.	Ready-to use reaction mix for PCR. Contains Taq DNA Polymerase, reaction buffer, dNTP mix (with dUTP instead of dTTP), and 10 mM MgCl2.	12 015 102 001	3 vials, 64 μl each
12 158 825 001	15 vials, 64 μl each
2	blue	LightCycler® DNA Master HybProbe, MgCl2 stock solution, 25 mM	To adjust MgCl2 concentration in the reaction mix.	12 015 102 001	1 vial, 1 ml
12 158 825 001	2 vials, 1 ml each
3	colorless	LightCycler® DNA Master HybProbe, Water, PCR Grade	To adjust the final reaction volume.	12 015 102 001	2 vials, 1 ml each
12 158 825 001	7 vials, 1 ml each

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

• Nuclease-free, aerosol-resistant pipette tips
• Pipettes with disposable, positive-displacement tips
• Sterile reaction tubes for preparing master mixes and dilutions and for synthesizing cDNA (if performing two-step RT-PCR)

• 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 (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": "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 providing 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 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.



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": "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 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.Fig. 1: Emission spectra of fluorescent dyes.
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.Fig. 2: Principle of the Residual Protein Trypsin ELISA.
3. After annealing, an increase in temperature leads to elongation and displacement of the probes.Fig. 2: Principle of the Residual Protein Liberase ELISA.
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.
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.

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

LightCycler^® 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.

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

Procedure	Assay Time [min]
Optional: dilution of template DNA	5
PCR Setup	15
LightCycler®Carousel-Based System PCR run (including Melting Curve)	25
Total Assay Time	45

", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "Easy-to-use reaction mix for PCR, using HybProbe probes with the LightCycler^® Carousel-Based System.", "Country": "XG", "Code": "Product Characteristics", "Name": "Product Characteristics" } ] } } ] }