{ "ProductData": { "ID": "RMD-3.5.8.1.1.3", "ProductType": "Others", "BrandName": "LightCycler^® 480 Genotyping Master", "ProductNameAddition": "", "RegulatoryDisclaimer1": "For general laboratory use.", "DisclaimerGroup1": "GLU", "RegulatoryDisclaimer2": null, "DisclaimerGroup2": null, "RegulatoryDisclaimer3": null, "SampleType": [ "Genomic DNA", "cDNA", "Plasmid DNA" ], "LicenseDisclaimers": [ ], "RelatedLinks": "", "Clone": "", "ControlTissue": [ "" ], "ISOtypes": "", "Species": [ "" ], "StainLocalization": [ "" ], "ProductNameGlobal": "LightCycler 480 Genotyping Master" }, "ProductImageDetails": { "ImagePath": "https://pim-media.roche.com/Images/Article_04707524001_im_en.png", "ImageType": "Image main" }, "Product2Taxonomy": { "Product2TaxonomyReferences": [ { "StructureSystemIdentifier": "Applications", "StructureSystemName": "Applications", "NodeID": "99-00-00", "StructureNodeStatus": "Inactive", "NodeName": "" }, { "StructureSystemIdentifier": "Pathogens", "StructureSystemName": "Pathogens", "NodeID": "99-00-00", "StructureNodeStatus": "Inactive", "NodeName": "" }, { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "NodeID": "06-0017", "StructureNodeStatus": "Active", "NodeName": "LightCycler 480 Kits" }, { "StructureSystemIdentifier": "OWP_Techniques", "StructureSystemName": "Techniques", "NodeID": "210-00", "StructureNodeStatus": "Active", "NodeName": "PCR/qPCR" }, { "StructureSystemIdentifier": "Lab_Type", "StructureSystemName": "Lab Types", "NodeID": "150-00", "StructureNodeStatus": "Active", "NodeName": "Research Lab" }, { "StructureSystemIdentifier": "Product_Solutions", "StructureSystemName": "Product Solutions", "NodeID": "100", "StructureNodeStatus": "Active", "NodeName": "Molecular" }, { "StructureSystemIdentifier": "Disease_Areas", "StructureSystemName": "Disease Areas", "NodeID": "99-00-00", "StructureNodeStatus": "Inactive", "NodeName": "" }, { "StructureSystemIdentifier": "OWP_Family", "StructureSystemName": "Product Families", "NodeID": "230", "StructureNodeStatus": "Active", "NodeName": "LightCycler" }, { "StructureSystemIdentifier": "Health_Topics", "StructureSystemName": "Health Topics", "NodeID": "99-00-00", "StructureNodeStatus": "Inactive", "NodeName": "" }, { "StructureSystemIdentifier": "Lab_Type", "StructureSystemName": "Lab Types", "NodeID": "170-00", "StructureNodeStatus": "Inactive", "NodeName": "" }, { "StructureSystemIdentifier": "OWP_Product_Types", "StructureSystemName": "Product Types", "NodeID": "20-000-00", "StructureNodeStatus": "Active", "NodeName": "Assays Reagents and Strips" }, { "StructureSystemIdentifier": "Lab_Type", "StructureSystemName": "Lab Types", "NodeID": "010-00", "StructureNodeStatus": "Active", "NodeName": "Molecular Lab" } ] }, "Product2Materials": { "P2MaterialReferences": [ { "MaterialNum": "04707524001", "MaterialDescription": "LightCycler 480 Genotyping Master", "RegisteredProductName": "LightCycler 480 Genotyping Master", "GTIN": "04038377021843", "ProductCategoryText": "Reagents, kits", "OldMaterialNumber": "", "PackSizePIM360": "4 × 384 μL", "PackSizeDescPIM360": "5x conc.
384 reactions of 20 μL final volume each", "MaterialAnnotation": "", "ReadyForUse": "false", "OrderInformation": "" } ] }, "Product2Products": { "Product2ProductReference": [ ] }, "ProductSpec": [ { "ProductSpecVariant": { "Chapters": [ { "Name": "Storage Conditions (Product)", "Value": "Store the kit at −15 to −25°C.", "Language": "en", "Country": "XG", "Code": "Storage Conditions (Product)" }, { "Name": "Sample Materials", "Value": "Use any template DNA suitable for qPCR, or cDNA suitable for PCR in terms of purity, concentration, and absence of PCR inhibitors.
For reproducible isolation of nucleic acids, use:

• Either a MagNA Pure System together with a dedicated reagent kit (for automated isolation),
• or a High Pure Nucleic Acid Isolation kit (for manual isolation).
• Use up to 50 ng complex genomic DNA or 1 to 10⁸ copies plasmid DNA for a 20 μL reaction. For larger volumes, the amount of template can be increased equivalently.

", "Language": "en", "Country": "XG", "Code": "Sample Materials" }, { "Name": "Product Characteristics", "Value": "Ready-to-use hot start reaction mix for PCR, followed by melting curve analysis for genotyping using the LightCycler^® PRO or LightCycler^® 480 Systems.", "Language": "en", "Country": "XG", "Code": "Product Characteristics" }, { "Name": "Product Description", "Value": "The LightCycler^® 480 Genotyping Master is a hot start reaction mix for PCR. The supplied enzyme contains a 5'-3'-exo-minus, N-terminal deletion of thermostable recombinant Taq DNA polymerase that is inactive at room temperature due to a chemical modification, and becomes activated during a 10-minute incubation at +95°C. HybProbe probes or SimpleProbe probes are used as detection format during PCR and subsequent melting curve analysis.

Since the mix is provided as a one-component, easy-to-use reagent, reaction setup only requires the addition of template DNA and primers. The mix can be used with different types of DNA (e.g., genomic, cDNA), and is ideally suited for high-throughput applications in 96- or 384-well plates.", "Language": "en", "Country": "XG", "Code": "Product Description" }, { "Name": "Applications", "Value": "The LightCycler^® 480 Genotyping Master is designed for use on the LightCycler^® PRO or LightCycler^® 480 System.
The kit contains a ready-to-use hot start reaction mix designed specifically for genotyping (SNP analysis with melting curves). It is optimized for use with HybProbe probes but can also be used with SimpleProbe probes.

The kit can also help prevent carryover contamination during PCR when used with LightCycler^® Uracil-DNA Glycosylase or to perform the second step of a two-step RT-PCR.", "Language": "en", "Country": "XG", "Code": "Applications" }, { "Name": "Control Reactions", "Value": "Always run appropriate positive and negative controls with the samples.

• To check for the presence of contamination, prepare and include a negative control by replacing the template DNA with Water, PCR Grade (Vial 2).
• For a 2-step RT-PCR setup, omit the addition of reverse transcriptase to the cDNA synthesis reaction; this will indicate whether DNA in RNA samples causes false-positive results.

", "Language": "en", "Country": "XG", "Code": "Control Reactions" }, { "Name": "Quality Control", "Value": "The LightCycler^® 480 Genotyping Master is function tested using the LightCycler^® System.", "Language": "en", "Country": "XG", "Code": "Quality Control" }, { "Name": "Additional Equipment and Reagent Required", "Value": "

• Nuclease-free pipette tips
• 1.5 mL RNase-free microcentrifuge tubes to prepare master mixes and dilutions
• To minimize risk of RNase contamination, autoclave all vessels

• Real-Time PCR systems such as the LightCycler^® PRO or LightCycler^® 480 Systems*
• LightCycler^® 480 Multiwell Plate 96, white*
• LightCycler^® 480 Multiwell Plate 384, white*
• LightCycler^® 480 Multiwell Plate 96, white, 4 bar codes*
• LightCycler^® 480 Multiwell Plate 384, white, 4 bar codes*
• Sealing Foil Applicator*
• LightCycler^® 480 Sealing Foil*
• LightCycler^® 8-Tube Strips (white)*
• LightCycler^® 8-Tube Strip Adapter Plate*
• Centrifuge with swinging-bucket rotor
• LightCycler^® Uracil-DNA Glycosylase* (optional)
  For details about prevention of carryover contamination, see Section, Prevention of Carryover Contamination.

", "Language": "en", "Country": "XG", "Code": "Additional Equipment and Reagent Required" }, { "Name": "Principle", "Value": "HybProbe probes consist of two different oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of PCR cycles. One probe is labeled at the 5' end with a suitable acceptor fluorophore (Red 610, Red 640, or Cy5), and to avoid extension, modified at the 3' end by phosphorylation. The second probe is labeled at the 3' end with the donor dye fluorescein (Fluos). Only after hybridization, the two probes are in close proximity, resulting in fluorescence resonance energy transfer (FRET) between the two fluorophores. During FRET, fluorescein, the donor fluorophore, is excited by the light source of the LightCycler^® System Instrument, and part of the excitation energy is transferred to a red acceptor fluorophore. The emitted fluorescence of the acceptor fluorophore is measured. Red 610, Red 640, or Cy5-labeled HybProbe probes can be used separately or in combination, therefore allowing single- or multiple-color detection.
The SimpleProbe format uses only one oligonucleotide probe. This single probe is designed to specifically hybridize to a target sequence that contains the SNP of interest. Once hybridized, the SimpleProbe probe emits much more fluorescence than it does when it is not hybridized. As a result, fluorescent signal changes are based solely on the hybridization status of the probe. SimpleProbe assays can distinguish between wild type, mutant, and heterozygous samples.The LightCycler^® 480 Genotyping Master is a ready-to-use reaction mix designed for the HybProbe probe detection format and SimpleProbe probe detection format using the LightCycler^® System. It is used to perform hot start PCR in LightCycler^® 480 Multiwell Plates.
Hot start PCR has been shown to significantly improve the specificity and sensitivity of PCR by minimizing the formation of nonspecific amplification products at the beginning of the reaction.
The polymerase in this master is a 5'-3'-exo-minus, N-terminal deletion of thermostable recombinant Taq DNA polymerase and furthermore, is chemically modified. It shows no activity up to +75°C because of the heat-labile blocking groups on some of the amino acid residues of the enzyme. Therefore, there is no elongation during nonspecific primer binding. The modified enzyme is “activated” by removing the blocking groups at a high temperature (that is, pre-incubation at +95°C for a maximum of 10 minutes).
The LightCycler^® 480 Genotyping Master provides convenience, excellent performance, reproducibility, and minimal contamination risk. All you have to supply is template DNA, PCR primers, HybProbe or SimpleProbe probes, and additional MgCl₂ (if necessary).", "Language": "en", "Country": "XG", "Code": "Principle" }, { "Name": "Content", "Value": "

Vial / Bottle	Cap	Label	Function/Description	Content
1	yellow	LightCycler® 480 Genotyping Master, 5x conc.	Ready-to-use hot start PCR reaction mix. Contains a modified Taq DNA Polymerase, reaction buffer, dNTP mix (with dUTP instead of dTTP), and 15 mM MgCl2.	4 vials, 384 μL each
2	colorless	LightCycler® 480 Genotyping Master, Water, PCR Grade	To adjust the final reaction volume.	4 vials, 1 mL each
3	blue	LightCycler® 480 Genotyping Master, MgCl2, 25 mM	To adjust MgCl2 concentration, if necessary.	1 vial, 1 mL

", "Language": "en", "Country": "XG", "Code": "Content" }, { "Name": "General Considerations", "Value": "In principle, the LightCycler^® 480 Genotyping Master can be used to amplify and detect any DNA target. However, you would need to adapt your detection protocol to the reaction conditions of the respective LightCycler^® Instrument, and design specific PCR primers and probes for each target. See the LightCycler^® PRO System User Assistance or the LightCycler^® 480 Instrument Operator's Manual for general recommendations.

The amplicon size should not exceed 700 bp in length. For optimal results, select a product length of 100 to 500 bp for monoplex and ≤350 bp for multiplex assays.

Detection formats

You can use this kit for both the HybProbe and SimpleProbe detection format.

Use a 1:5 dilution of the LightCycler^® 480 Genotyping Master to prepare the PCR Mix for HybProbe probes. The MgCl₂ concentration in this mix gives optimal results with nearly all primer combinations.

Use a 1:10 dilution of the LightCycler^® 480 Genotyping Master to prepare the PCR Mix for SimpleProbe probes. For this type of analysis, it might be helpful to add additional MgCl₂ (Vial 3). Melting curve results will improve if you dilute the mix even further, for example, using a 1:20 dilution. If you dilute the mix further, you must also add additional MgCl₂ to the reaction. The final concentration must be 3 mM, therefore, add an extra 1.8 μL MgCl₂ from Vial 3 if you dilute 1:20.

Two-Step RT-PCR

LightCycler^® 480 Genotyping Master can also be used to perform two-step RT-PCR. In two-step RT-PCR, the first step, reverse transcription of RNA into cDNA, is performed outside the LightCycler^® System. Subsequent amplification and online monitoring is performed according to a standard LightCycler^® System procedure, using cDNA as starting sample material.
One of the following reagents is required for reverse transcription of RNA into cDNA (see Ordering Information section for details):

• Transcriptor Reverse Transcriptase*
• Transcriptor First Strand cDNA Synthesis Kit*

Synthesis of cDNA is performed according to the detailed instructions provided with the cDNA synthesis reagent.

For initial experiments, run undiluted, 1:10 diluted, and 1:100 diluted cDNA template in parallel to determine the optimum template amount.

", "Language": "en", "Country": "XG", "Code": "General Considerations" }, { "Name": "Product Purpose", "Value": "The LightCycler^® 480 Genotyping Master is a hot start reaction mix for PCR. The supplied enzyme contains a 5'-3'-exo-minus, N-terminal deletion of thermostable recombinant Taq DNA polymerase that is inactive at room temperature due to a chemical modification, and becomes activated during a 10-minute incubation at +95°C. HybProbe probes or SimpleProbe probes are used as detection format during PCR and subsequent melting curve analysis.

Since the mix is provided as a one-component, easy-to-use reagent, reaction setup only requires the addition of template DNA and primers. The mix can be used with different types of DNA (e.g., genomic, cDNA), and is ideally suited for high-throughput applications in 96- or 384-well plates.", "Language": "en", "Country": "XG", "Code": "Product Purpose" } ] } } ] }