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• LightCycler^® 2.0 Instrument
• LightCycler^® Capillaries
• LightCycler^® Centrifuge Adapters (included with the LightCycler^® Carousel-Based System) and a 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 Carousels
  or
• LC Carousel Centrifuge and LC Carousel Centrifuge 2.0 Rotor Set, to centrifuge the LightCycler^® 2.0 Sample Carousels in the LC Carousel Centrifuge
• LightCycler^® Probe Design Software 2.0
• Real-Time PCR using HybProbe Probes:
  LightCycler^® Control Kit DNA
  LightCycler^® Color Compensation Set
  LightCycler^® DNA Master HybProbe
  LightCycler^® FastStart DNA Master HybProbe
  LightCycler^® FastStart DNA Master^PLUS HybProbe
  LightCycler^® Multiplex DNA Master HybProbe
• Real-time PCR using SYBR Green I:
  LightCycler^® DNA Master SYBR Green I
  LightCycler^® FastStart DNA Master SYBR Green I
  LightCycler^® FastStart DNA Master^PLUS SYBR Green I
• Real-time PCR using Hydrolysis Probes:
  LightCycler^® Taqman^® Master
• Real-time RT-PCR using on HybProbe Probes or SYBR Green I:
  LightCycler^® Control Kit RNA
  LightCycler^® RNA Amplification Kit HybProbe
  LightCycler^® RNA Master HybProbe
  LightCycler^® RNA Amplification Kit SYBR Green I
  LightCycler^® RNA Master SYBR Green I
• LightCycler^® Uracil-DNA Glycosylase (for prevention of carry-over contamination)
• PCR template (genomic DNA or cDNA) or RT-PCR template (total RNA, mRNA, or cRNA)
• (RT-)PCR primers (easiest when designed for amplicons between 50 to 300 bp)
• HybProbe probes (for single, or multi-color applications: LightCycler^® Red 610, LightCycler^® Red 640, Cy5 {670}, or Cy5.5 {705})
• Hydrolysis probes (for single, or multi-color applications)
• Nuclease-free, aerosol-resistant pipette tips
• Pipettes with disposable, positive-displacement tips
• Sterile reaction (Eppendorf) tubes for preparing master mixes and dilutions

", "Name": "Additional Equipment and Reagent Required" }, { "Language": "en", "Value": "

\n\t
1. LightCycler^® 2.0 Instrument 
\n\t
2. LightCycler^® 2.0 Instrument Operator's Manual
\n\t
3. LightCycler^® 2.0 Sample Carousel (20 μl)
\n\t
4. LightCycler^® Software 4.1 CD-ROM
\n\t
5. LightCycler^® Software 4.05 Tutorial CD-ROM
\n\t
6. LightCycler^® Centrifuge Adapters with Cooling Block
\n\t
7. LightCycler^® Capping Tool
\n\t
8. LightCycler^® 2.0 Capillary Releaser
\n\t
9. Serial data cable (RS 232)
\n\t
10. Power mains cable (US and EU)
\n\t
11. Identification caps (Set of 2)
\n\t
12. LightCycler Sample Carousel 0-Ring (Set of 2)
\n\t
13. Brushes (4 pieces)
\n

\n
\n ", "Name": "Content" }, { "Language": "en", "Value": "The LightCycler^® 2.0 Instrument enables the monitoring of amplification of 32 PCR products simultaneously, in real-time and online, with six different detection channels. The LightCycler^® Software 4.1 collects fluorescence data from all six channels at each cycle and displays it immediately.
Amplification occurs in specially designed reaction capillaries. Their high surface-to-volume ratio provides very rapid thermal transfer, which facilitates high-speed thermal cycling.
The combination of LightCycler^® Capillaries and the air-driven temperature control of the LightCycler^® 2.0 Instrument ensures rapid PCR. An entire 35-cycle run can be performed in as little as 30 minutes (with 20 μl capillaries) to 60 minutes (with 100 μl capillaries).
Fluorescence detection formats: The LightCycler^® 2.0 Instrument is optimized for three fluorescence detection formats: SYBR Green I, HybProbe probes and hydrolysis probes. In addition, the instrument supports a wide variety of other fluorescence detection formats, such as monocolor SimpleProbe probes and other formats based on FRET (fluorescence resonance energy transfer).
Software: The LightCycler^® 2.0 Instrument utilizes the LightCycler^® Software 4.1, an optimized version of the LightCycler^® 4.0 and 4.05 Software, which provides advanced data analysis modules, efficient data management and effective data protection.", "Name": "Product Description" }, { "Language": "en", "Value": "Fluorescence readings during cycling

For SYBR Green I, take the reading at the end of Elongation. In the LightCycler^® Software, the Acquisition Mode should be ‘Single’ in the Elongation step.

For HybProbe Probes, take the reading at the end of Annealing. In the LightCycler^® Software, the Acquisition Mode should be ‘Single’ in the Annealing step.

For Hydrolysis Probes, take the reading at the end of Annealing/Elongation. In the LightCycler^® Software, the Acquisition Mode should be ‘Single’ in the Annealing/Elongation step.

For Molecular Beacons, take the reading at the end of Annealing. In the LightCycler^® Software, the Acquisition Mode should be ‘Single’ in the Annealing step.

For Scorpion Probes, take the reading 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 amplification analysis of SYBR Green I or other probe formats or Melting Curve analysis of SYBR Green I or other probe formats, 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.", "Name": "Protocols - Help Corner" }, { "Language": "en", "Value": "Borosilicate glass 20 μl or 100 μl LightCycler^® Capillaries filled with total RNA, mRNA or DNA templates, in combination with the appropriate reaction mix.
Note: The glass capillaries can only be used with their dedicated sample carousel, namely the LightCycler^® 2.0 Sample Carousel (20 μl) for 20 μl glass capillaries, or the LightCycler^® 2.0 Sample Carousel (100 μl) for 100 μl glass capillaries.", "Name": "Sample Materials" }, { "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 so called \"seek threshold\") to be recognized by the instrument. With both HybProbe probes and SYBR Green I, 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 and SYBR Green I reagents, keep them away from light!



Cleaning of the LightCycler^® 2.0 Instrument

Clean the housing of the LightCycler^® 2.0 Instrument with a mild commercial detergent. Alternatively, 70 % ethanol can be used for cleaning the instrument housing.

If any capillaries broke during a run, clean the thermal chamber of the instrument with a soft lint-free cloth (e.g., Kimberley Clark) moistened with decontamination solution. This can be 70% Mikrozid (Schülke & Mayr GmbH, Norderstedt, Germany) or 1:3 Clorox Regular (The Clorox Company, Oakland, USA), or 3% Kohrsolin (Bode Chemie GmbH, Hamburg, Germany), or 70% Ethanol (this only cleans, no decontamination).



Manually opening the LightCycler^® 2.0 Instrument

The lid lock is regulated electronically. For manual opening (e.g., if no electricity is available), the lid can be unlocked manually, by pressing the hidden button under the front left side of the instrument.

Note: If the instrument is unlocked manually during a run, the run will be aborted and all the data will be lost.", "Name": "Troubleshooting - Help Corner" }, { "Language": "en", "Value": "Use the LightCycler^® 2.0 Instrument to perform:

• Fast and sensitive real-time PCR and RT-PCR
• Reproducible and reliable kinetic quantification of DNA, cDNA, or RNA
• Easy PCR product identification and characterization by melting point analysis
• Highly specific mutation analysis and SNP (Single Nucleotide Polymorphism) genotyping

", "Name": "Applications" }, { "Language": "en", "Value": "Detection Sensitivity of the LightCycler^® Carousel-Based System
The detection sensitivity of the LightCycler^® Carousel-Based System has been tested with a number of target sequences in human genomic DNA, plasmid DNA, total liver RNA and in vitro transcribed RNA. Sensitivity depends on the target and optimization of the reaction, as in every PCR. Detection of one copy of a target has been demonstrated in several publications.

For SYBR Green I format, 3 pg of human genomic DNA^# or 1 plasmid copy have been detected with PCR.

For the format of the HybProbe Probe, 3 pg of human genomic DNA^# or 1 plasmid copy have been detected with PCR.

For SYBR Green I format, 1 pg of total liver RNA* or 50 RNA copies have been detected with one-step RT-PCR.

For the format of the HybProbe Probe, 1 pg of total liver RNA* or 10 RNA copies have been detected with one-step RT-PCR.

For SYBR Green I format, 0.1 pg of total RNA* or 5 mRNA copies have been detected with two-step RT-PCR.

For the format of the HybProbe Probe, 0.5 pg of total RNA* or 5 mRNA copies have been detected with two-step RT-PCR.

Note: ^#Three pg of human genomic DNA corresponds to one genome equivalent.
Note: *Fifty pg of total liver RNA corresponds to one cell.


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^® reagents.


Recommended HybProbe Probes for the LightCycler^® Carousel-Based System
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 [610], LightCycler^® Red 640 [640], Cy5 [670] and Cy5.5 [705].


Recommended Dyes for the LightCycler^® Carousel-Based System
The optical unit of the LightCycler^® 2.0 Instrument can measure fluorescence in six separate channels, simultaneously. The different channels are optimized for the following fluorescent dyes:

• Channel 530: measures at 530 nm (+/- 10 nm) and is optimized for SYBR Green I, Fluorescein and FAM
• Channel 560: measures at 555 nm (+/- 10 nm) and is optimized for HEX/VIC
• Channel 610: measures at 610 nm (+/- 10 nm) and is optimized for LightCycler^® Red 610
• Channel 640: measures at 640 nm (+/- 10 nm) and is optimized for LightCycler^® Red 640
• Channel 670: measures at 670 nm (+/- 10 nm) and is optimized for Cy5
• Channel 705: measures at 710 nm (+/- 10 nm) and is optimized for Cy5.5

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.", "Name": "Background Information - Help Corner" } ] } } ] }