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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": "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 multicolor experiments. Due to the presence of several Fluorescein labeled probes in dual and multicolor 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 multicolor 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^® FastStart DNA Master^PLUS 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 Tm 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^® FastStart DNA Master^PLUS 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": "

Procedure	Assay Time 20 μl reactions [min]	Assay Time 100 μl reactions [min]
PCR Setup	15	15
LightCycler® Carousel-Based System PCR run	45	90
Total Assay Time	60	105

", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "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, LightCycler^® Red 640, Cy5, or Cy5.5) and to avoid extension, modified at the 3' end by phosphorylation. 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.LightCycler^® FastStart DNA Master^PLUS HybProbe is a ready-to-use reaction mix designed specifically for the HybProbe probes detection format using the LightCycler^® Carousel-Based System. It is used to perform hot start PCR in glass capillaries. Hot start PCR has been shown to significantly improve the specificity and sensitivity of PCR (Chou, Q., et al., 1992; Kellogg, D.E., et al., 1994; Birch, D.E., et al., 1996), by minimizing the formation of nonspecific amplification products at the beginning of the reaction.
FastStart Taq DNA Polymerase is a modified form of thermostable recombinant Taq DNA polymerase that is inactive at room temperature and below. The enzyme is active only at high temperatures, where primers no longer bind nonspecifically. The enzyme is 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^PLUS HybProbe provides convenience, excellent performance, reproducibility, and minimal contamination risk. All you have to supply is template DNA, PCR primers, and HybProbe probes.
In combination with the LightCycler^® Carousel-Based System and suitable PCR primers, this kit allows very sensitive detection and quantification of defined DNA sequences. Various sources of DNA (cDNA, genomic DNA, plasmid DNA, etc.) can be used. The kit is also suitable for SNP and mutation detection using melting curve analysis. Furthermore, the kit can be used to perform two-step RT-PCR in combination with a reverse transcription kit for cDNA synthesis. 

", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "If precipitates are seen in the LightCycler^® FastStart DNA Master^PLUS HybProbe, place the vial at +37°C and mix gently from time to time, until the precipitate is completely dissolved. This treatment does not influence the performance of PCR. Generally, precipitates should not be seen, as the buffer of the LightCycler^® FastStart DNA Master^PLUS HybProbe was reformulated and optimized after the observation of precipitates in the LightCycler^® FastStart DNA Master HybProbe.



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": "

Vial / Bottle	Cap	Label	Function / Description	Catalog Number	Content
1a	white	LC FastStart DNA MasterPLUS HybProbe, Enzyme	Contains FastStart Taq DNA Polymerase, reaction buffer, MgCl2, and dNTP mix (with dUTP instead of dTTP).	Ready-to-use hot start PCR Master after pipetting 10 μl from Vial 1a into one Vial 1b.	03 515 575 001	1 vial 1a and 3 vials 1b, for 3 × 128 μl each LC FastStart DNA MasterPLUS HybProbe, 5x conc.
03 515 567 001	5 vials 1a and 15 vials 1b, for 15 × 128 μl each LC FastStart DNA MasterPLUS HybProbe, 5x conc.
1b	red	LC FastStart DNA MasterPLUS HybProbe, Reaction Mix	Ready-to-use hot start PCR Master after pipetting 50 μl from Vial 1a into one Vial 1b.	03 752 178 001	4 vials 1a and 12 vials 1b, for 12 × 640 μl each LC FastStart DNA MasterPLUS HybProbe, 5x conc.
2	colorless	LC FastStart DNA MasterPLUS HybProbe, Water, PCR Grade	To adjust the final reaction volume.	03 515 575 001	2 vials, 1 ml each
03 515 567 001	7 vials, 1 ml each
03 752 178 001	2 vials, 25 ml each

", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "LightCycler^® FastStart DNA Master^PLUS HybProbe is designed for PCR applications using the LightCycler^® Carousel-Based System. The kit is ideally suited for hot start PCR applications.
The kit can also be used in conjunction with heat-labile Uracil-DNA Glycosylase to prevent carryover contamination during PCR.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "

• Nuclease-free, aerosol-resistant pipette tips
• Pipettes with disposable, positive-displacement tips
• Sterile reaction tubes for preparing master mixes and dilutions

• LightCycler^® Carousel-Based System*
• LightCycler^® Capillaries*

• Standard benchtop microcentrifuge containing a rotor for 2 ml reaction tubes

or

• LC Carousel Centrifuge 2.0* for use with the LightCycler^® 2.0 Sample Carousel (optional)

• LightCycler^® Uracil-DNA Glycosylase* (optional)

• LightCycler^® Color Compensation Set* (optional)

", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" } ] } } ] }