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Starting with serial number 29001 for the LightCycle® 480 Instrument II 96-well version (Cat. No. 05 015 278 001), and serial number 5631 for LightCycler® 480 Instrument II 384-well version (Cat. No. 05 015 243 001), a high power, broad spectra LED (Light Emitting Diode) with long lifetime is replacing the Xenon Lamp.
The white power LED is the successor of the Xenon Lamp and has the same functionality. Data generated on a Xenon Lamp instrument and on a LED instrument are fully comparable.
For LightCycler® 480 Instruments with an LED, the lamp unit and the liquid light guide are no longer used since the LED lamp is directly mounted to the Optics unit. The average lifetime of the LED is >10,000 hours and therefore a lamp exchange by the customer is not required. Replacing the LED is no longer the responsibility of the customer and therefore, not provided as an accessory. If the LED does break, please contact your local Roche representative (LED can only be exchanged by a service engineer).
Universal CC keys can be applied to LightCycler® 480 Instrument II with Xenon Lamp as well as with LED.
Definition of the LightCycler® 480 Instrument Fluorescein Detection Limit
The optical system of the LightCycler® 480 Instrument has a lower limit of detection of 0.1 nmol/l Fluorescein, which corresponds to 2 fmol or 1.2 x 109 Fluorescein Molecules in a 20 μl reaction. The lower detection limit is defined as the concentration of Fluorescein required to generated a signal which is significantly (threefold standard deviation) above system background noise (defined by a buffer-filled plate). Important: The lower detection limit is not related to PCR sensitivity!
Reduction of Background Fluorescence in the LightCycler® 480 Instrument
The optical system of the LightCycler® 480 Instrument uses dye-specific excitation and detection filters. Using an excitation filter ensures that only light of the wavelength required to excite the desired fluorescent dye reaches the multiwell plate. Other dyes in the reaction are not excited. Using a detection filter specific for the emitted fluorescence light ensures unwanted background fluorescence is not measured. The LightCycler® 480 Instrument excites and detects different dyes in the reaction in succession by applying different filter combinations, thus minimizing generation and detection of background fluorescence.
No Need for a Reference Channel
The optical system of the LightCycler® 480 Instrument uses dye-specific excitation, minimizing unwanted background fluorescence. Calculations using a reference channel is not required. The AbsQuant analysis module of the LightCycler® 480 Software automatically performs arithmetic background correction.
Good reasons to choose a Xenon Lamp instead of a Halogen Lamp as a light source for the LightCycler® 480 Instrument
The main difference between a Xenon and a Halogen lamp is their luminous intensity, which differs at least by factor 10. While a 100 W Halogen lamp typically has an intensity around 0.8 μW/mm2 at a wavelength of 483 nm, a 100 W Xenon lamp has an intensity of 10 μW/mm2. At a wavelength of 450 nm the luminous intensity of the two lamps even differs by factor 20. In particular for the HybProbe detection format, the lower intensity of the halogen lamp is not sufficient for achieving adequate excitation of long-wavelength reporters such as Cy5. The halogen lamp's wavelength range of emitted light is however comparable (420 nm - 670 nm).
No new color compensation keys necessary after an exchange of the Xenon Lamp
It is not necessary to create new color compensation keys after a Xenon Lamp exchange, because color compensation is independent of light intensity. In addition, any possible wavelength shifts are compensated by the excitation filters.
Light Pipe and Liquid Light Guide in the LightCycler® 480 Instrument
In the LightCycler® 480 Instrument, the light pipe consists of a rectangular glass bar. Light is transmitted from the emission light source (the Xenon lamp) to the optical unit through this glass bar by total reflectance; thereby the light is also homogenized. The light pipe converts the circular profile of the light guide into a rectangular profile by multiple rounds of reflection, which matches the profile of the multiwell plate, resulting in homogeneous illumination of the multiwell plate.
The purpose of the liquid light guide is to conduct the flow of light from the emission light source (the Xenon lamp module) to the optics module. The major advantage of using light guides is their higher transmission compared to glass optical fibers.
Resolution for an Image of a 384-well Multiwell Plate
Approximately 400 pixels are used to image a single well of a 384-well multiwell plate. The resolution of the optical system is also sufficient for LightCycler® 1536 Multiwell Plates.
Measurement of Additional Dyes
In addition to the dyes listed in the LightCycler® 480 Instrument Operator's Manual, all other dyes that are compatible with the excitation and emission filter wavelengths can be measured by the LightCycler® 480 Instrument.
Using Molecular Beacons in Multicolor Applications
Molecular Beacons should work in multiplex reactions performed using LightCycler® 480 Instruments. Please note however, that using FAM and TET together in a single reaction is not possible. The optimal excitation wavelength for FAM is 494 nm and for TET 521 nm. The optimal emission wavelength for FAM is 518 nm and for TET 536 nm. The high similarity of these two spectra increases the risk of crosstalk. A suitable dye combination is, e.g., FAM/Cy-5. Please refer to the LightCycler® 480 Instrument Operator's Manual to find more suitable dye combinations for multicolor applications.
THERMAL BLOCK CYCLER
The expected life time of the thermal block cycler in the LightCycler® 480 Instrument
The guaranteed minimum lifetime of the thermal block cycler is 105,000 cycles (corresponding to approx. 4 runs with 43 cycles on 200 days over 3 years).
No Recalibration of the LightCycler® 480 Instrument after exchange of the thermal block cycler unit
The reason therefore is, that each thermal block cycler unit has its own EEPROM*, carrying block-specific information (like calibration-data). During start-up of the instrument the information within the EEPROM is read by the instrument controller which transfers it to the LightCycler® 480 Software. (*EEPROM = Electrically Erasable Programmable Read-Only Memory)
Temperature Homogeneity
The thermal block cycler of the LightCycler® 480 Instrument has three different temperature sensors: one sensor is located in the center of the plate mount and is used for absolute temperature control, while the other two sensors are located on top of the left and right section of the block and are used to check temperature homogeneity. Failure of specified absolute temperature and of homogeneity produces an error message.
General Cleaning of the Thermal Block Cycler
Regular cleaning of the LightCycler® 480 Instruments and accessories is not required. When necessary, clean the housing of the LightCycler® 480 Instrument, the thermal block cycler, and the block cycler cover with a mild commercial detergent. Use 70% ethanol for disinfecting the instrument housing, the thermal block cycler, and the block cycler cover.
Cleaning of the LightCycler® 480 block cycler unit: pipette into all wells 20μl (384-well block) respectively 125μl (96 well block) of 70% ethanol or isopropanol. After waiting 15 minutes, pipette up and down several times. Remove the liquid and let the block cycler unit dry before using again. Take care not to degrade the instrument block coating.
LIMS
The meaning of the two LAN interfaces of the LightCycler® 480 Instrument control unit
Use the RJ 45 port located next to the COM 2 port to connect the control unit to the LightCycler® 480 Instrument. Please note that this must be a one-to-one (peer-to-peer) connection. It is not possible to make this connection via a network hub. Use the provided LAN cable for this connection. Use the other LAN connector (Laboratory LAN) to interface the LightCycler® 480 System to a laboratory network by using the functions of the LightCycler® 480 LIMS Interface Module, including starting an experiment, obtaining the experiment's status, reading the multiwell plate bar-code, receiving results, etc. A detailed list of all functions of the LightCycler® 480 LIMS Interface Module can be found in the corresponding LightCycler® 480 Instrument Operator's Guide. Note: To connect several LightCycler® 480 Instruments to a laboratory network you do need a control unit for each instrument, because (as mentioned above) the connection between instrument and control unit must always be a direct connection. Please note that Roche does not support installations with a network hub between instrument and control unit.
CONSUMABLES
Use of Strip PCR Tubes
A LightCycler® 8-Tube Strip Adapter Plate (Cat. No. 06 612 598 001) is available for use with the LightCycler® 480 System and LightCycler® 8-Tube Strips. The adapter plate can hold up to 12 LightCycler® 8-Tube Strips in one run and can be used multiple times. The LightCycler® 8-Tube Strip Adapter Plate can only be used with the LightCycler® 480 Instrument I and LightCycler® 480 Instrument II, 96-well version.
Universal ProbeLibrary Assays and the Appropriate Master Mix on Your Real-Time PCR Instrument
Universal ProbeLibrary assays designed with ProbeFinder Software can be performed on any real-time PCR instrument without further optimization of assay conditions. Universal ProbeLibrary assays are compatible with all real-time PCR instruments capable of detecting fluorescein, FITC, FAM and/or SYBR Green I. Use a standard real-time PCR protocol for hydrolysis probes, taking into account recommendations in the supplier's package insert for each respective master mix.
For use in combination with the LightCycler® 480 Instrument: LightCycler® 480 Instrument 96 or 384 LightCycler® 480 Probes Master LightCycler® 480 Multiwell Plate 96* or LightCycler® 480 Multiwell Plate 384 LightCycler® 480 Sealing Foil Standard swing-bucket centrifuge containing a rotor and adapters for multiwell plates",
"Country": "XG",
"Code": "Background Information - Help Corner",
"Name": "Background Information - Help Corner"
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"Language": "en",
"Value": "
LightCycler® 480 Instrument, 96- or 384-well
LightCycler® 480 Block Cycler Unit, 96- or 384-well
LightCycler® 480 Software*
LightCycler® 480 Instrument Operator’s Manual
LightCycler® 480 Xenon Lamp (Spare Lamp)
Pentium Desktop PC
* including absolute quantification, Tm calling, relative quantification, melting curve-based genotyping, and endpoint genotyping.",
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"Value": "The LightCycler® 480 Instrument is intended for performing rapid, accurate polymerase chain reaction (PCR) in combination with real-time, online detection, enabling Absolute or Relative Quantification of a target nucleic acid, as well as post-PCR analysis of the amplified nucleic acid by Melting Curve analysis.
The LightCycler® 480 Instrument is intended for life science research only and must be used exclusively by laboratory professionals trained in laboratory techniques and having studied the Instructions for Use of this instrument. The LightCycler® 480 Instrument is not for use in diagnostic procedures.",
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"Code": "Intended Use",
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