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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

", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "Variable, depending on the number of cycles, annealing time, and melting curve program. For example, a LightCycler^® 480 PCR run will last about 50 minutes if the program specifies 10 minutes pre-incubation, followed by 40 cycles amplification (each with 5 s denaturation, 5 s annealing, and 5 s elongation), and a monocolor melting curve analysis (see Results section).", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "The LightCycler^® 480 Genotyping Master is specifically adapted for multiplex real-time PCR followed by melting curve analysis on the LightCycler^® 480 Instrument. It is compatible with multiple HybProbe probes or SimpleProbe probes labeled with dyes supported by the LightCycler^® 480 Instrument for the detection of SNP variants. For optimal results, white LightCycler^® 480 Multiwell plates are recommended.


HybProbe Probes

The LightCycler^® Probe Design Software 2.0 can design the best HybProbe probe and primer combinations and give precise estimations of the Tms of the primers and probes in LightCycler^® reagents.

Performing a Melting Curve analysis with HybProbe probes can identify and characterize PCR product/probe hybrids using their specific melting behavior for genotyping and mutation analysis.", "Country": "XG", "Code": "Background Information - Help Corner", "Name": "Background Information - Help Corner" }, { "Language": "en", "Value": "The LightCycler^® 480 Genotyping Master is function tested using the LightCycler^® System.", "Country": "XG", "Code": "Quality Control", "Name": "Quality Control" }, { "Language": "en", "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 LightCycler^® 480 Instrument, and design specific PCR primers and probes for each target. See the LightCycler^® 480 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.

The performance of the kit described in this Instructions for Use is warranted only when it is used with the LightCycler^® 480 System.

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^® 480 System. Subsequent amplification and online monitoring is performed according to the standard LightCycler^® 480 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 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.

", "Country": "XG", "Code": "General Considerations", "Name": "General Considerations" }, { "Language": "en", "Value": "Use any template DNA, such as genomic or plasmid DNA, cDNA suitable for PCR, as long as it is sufficiently pure, concentrated, and free 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.

When using unpurified cDNA from a reverse transcription reaction, especially when it contains high concentrations of RNA and oligonucleotides, you can improve your results by using 2 μl (or less) of that sample in the reaction.

", "Country": "XG", "Code": "Sample Materials", "Name": "Sample Materials" }, { "Language": "en", "Value": "

Instruments and consumables

• LightCycler^® 480 Instrument II*
• LightCycler^® 480 Multiwell Plate 384* or LightCycler^® 480 Multiwell Plate 96*, white
• LightCycler^® 8-Tube Strip Adapter Plate*
• LightCycler^® 8-Tube Strips (white)*
• Standard swinging-bucket centrifuge containing a rotor for multiwell plates with suitable adaptors
• Nuclease free, aerosol-resistant pipette tips
• Pipettes with disposable, positive-displacement tips
• Sterile 1.5 ml reaction tubes for preparing master mixes and dilutions

Reagents for the LightCycler^® 480 Instrument II*

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

For details about prevention of carryover contamination, see the Prevention of Carryover Contamination section.

", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" }, { "Language": "en", "Value": "The LightCycler^® 480 Genotyping Master is designed for research studies on the LightCycler^® 480 System. The LightCycler^® 480 Genotyping Master is 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.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "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 (LightCycler^® Red 610, LightCycler^® 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^® 480 Instrument, and part of the excitation energy is transferred to LightCycler^® Red, the acceptor fluorophore. The emitted fluorescence of the acceptor fluorophore is measured.  LightCycler^® Red 610, LightCycler^® Red 640, or Cy5-labeled HybProbe probes can be used separately or in combination, therefore allowing single- or multiple-color detection. Refer to the LightCycler^® 480 Operator`s Manual for additional information.

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^® 480 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 (Chou, Q., et al., 1992, Kellogg D. E., et al., 1994, Birch, D. E., 1996) 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).", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "Always run a negative control with the samples. To prepare a negative control:

• Replace the template DNA with Water, PCR Grade (Vial 2). This will reveal whether a contamination problem exists.
• 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.

", "Country": "XG", "Code": "Control Reactions", "Name": "Control Reactions" }, { "Language": "en", "Value": "Ready-to-use hot start reaction mix for PCR, followed by melting curve analysis for genotyping using the LightCycler^® 480 System.", "Country": "XG", "Code": "Product Characteristics", "Name": "Product Characteristics" }, { "Language": "en", "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.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" } ] } } ] }