For life science research only. Not for use in diagnostic procedures. Others LightCycler 480 Control Kit LSR LightCycler® 480 Control Kit 3.5.8.3.6.0 04710924001 LightCycler 480 Control Kit LightCycler 480 Control Kit 04038377023687 Reagents, kits 1 kit 3 control reactions How this Product WorksExperiment A, QuantificationA 136 bp fragment of the human Cyp2C9 gene is amplified from plasmid DNA and detected with a short hydrolysis probe that is labeled with FAM (probe #80 from the Universal ProbeLibrary*). To test the precision of the LightCycler® 480 System, replicates with only 1,000 or 2,000 copies of target DNA per well are distributed throughout the plate and quantified with reference to a row of standards.As an internal control (to prove absence of PCR inhibition), a small amount (approx. 100 copies) of an artificial DNA template is added to each well. This control is co-amplified with the target DNA. Its amplification is detected simultaneously with a LightCycler® Red 610-labeled hydrolysis probe. The results are displayed in a separate optical channel. The distances between the wavelengths of the two detection channels (483 – 533 and 558 – 610, LightCycler® 480 Instrument I; 465 – 510 and 533 – 610, LightCycler® 480 Instrument II) are high enough that there is no need to use color compensation to correct for crosstalk.Alternatively, the target amplification can be detected using SYBR Green I. By subsequent melting curve analysis of the PCR product, the specificity of the reaction can be proven.Experiment B, GenotypingThe same 136 bp fragment of the CyP2C9 gene is amplified from different samples of plasmid DNA. This gene is known to contain a single nucleotide polymorphism (SNP), and various samples included in the experiment contain the wild type sequence, the homozygous point mutation, and heterozygote DNA with wild type and mutant strands. With HybProbe probes for detection, a subsequent melting curve analysis can be used for identification of the different genotypes, because the probe melts off the perfectly matched sequence and the mismatched sequence at different melting temperatures. en The LightCycler® 480 Control Kit is designed to prove the performance of all components of the LightCycler® 480 System, including instrument, software, consumables, generic reagents, and optional devices such as a pipetting robot. The kit is primarily for use with the LC 480 Multiplex DNA Master (vial 15), or alternatively with the LC 480 SYBR® Green I Master (vial 16) for Procedures A, but it can also be used with the LC 480 Genotyping Master (vial 14) for Procedure B.The test includes two control experiments. Experiment A is for absolute quantification of prediluted standard DNA. Experiment B is used for genotyping samples with a wild type DNA sequence as well as samples with a homozygous or heterozygous point mutation.The performance of the kit shown in this Instructions for Use is guaranteed only when it is used with the LightCycler® 480 System. en The LightCycler® 480 Control Kit is designed as a tool to confirm real-time PCR and melting curve analysis performance of the LightCycler® 480 Instrument. The kit can be used to verify the LightCycler® 480 System’s speed, accuracy, and intra-well assay reproducibility. SNP analysis with HybProbe probes and quantification with hydrolysis probes in a dual-color setup can both be demonstrated. en