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"Language": "en", "Value": "Store the kit at −15 to −25°C.", "Country": "XG", "Code": "Storage Conditions (Product)", "Name": "Storage Conditions (Product)" }, { "Language": "en", "Value": "LightCycler^® RNA Master SYBR Green I is designed for use in research. When combined with
the LightCycler^® Carousel-Based System, this kit uses a hot start RT-PCR protocol to provide very sensitive detection and quantification of defined RNA sequences (if suitable RT-PCR primers are supplied). The kit is especially suitable for difficult RNA populations, as the elevated incubation temperature during the reverse transcription step will help to overcome secondary structures. The hot start feature will minimize mispriming during the initial phase of the reaction and therefore overall sensitivity of RT-PCR is increased.

In addition, the kit can be used with Uracil-DNA Glycosylase, heat-labile to prevent carryover contamination during PCR.", "Country": "XG", "Code": "Applications (IFU)", "Name": "Applications (IFU)" }, { "Language": "en", "Value": "Easy-to-use SYBR Green I reaction mix for one-step RT-PCR, using the LightCycler^® Carousel-Based System.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "Roche recommends the LightCycler^® Probe Design Software 2.0 for designing RT-PCR primers. This software provides precise estimations of the T_ms of primers used with LightCycler^® Reagent Kits.



Performing a Melting Curve analysis with SYBR Green I can identify and characterize PCR products based on their specific melting behavior. Moreover, non-specific products (primer-dimers) can also be identified using this method.



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.", "Country": "XG", "Code": "Background Information - Help Corner", "Name": "Background Information - Help Corner" }, { "Language": "en", "Value": "

• Nuclease-free, aerosol-resistant pipette tips

• Sterile reaction tubes for preparing master mixes and dilutions

• LightCycler^® Carousel-Based System*
• LightCycler^® Capillaries*
• 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 Carousel (20 μl; optional)
• Uracil-DNA Glycosylase, heat-labile* (optional)
  For prevention of carryover contamination; see section Prevention of Carryover Contamination.

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

Procedure	Time [min]
RT-PCR Setup	15
Reverse Transcription	20
LightCycler® Carousel-Based System PCR run	25
Total Assay Time	60

", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "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 \"seek threshold\") to be recognized by the instrument. With 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 SYBR Green I dye, keep the LightCycler^® RNA Master SYBR Green I away from light!", "Country": "XG", "Code": "Troubleshooting - Help Corner", "Name": "Troubleshooting - Help Corner" }, { "Language": "en", "Value": "During cycling, Fluorescence readings for SYBR Green I should be taken at the end of elongation. In the LightCycler^® Software, the Acquisition Mode should be ‘Single’ in the Elongation/Extension 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

For both online display and analysis of SYBR Green I, the channel setting should be 530.

For further information regarding Channel Settings, please refer to the LightCycler^® Carousel-Based System LightCycler^® Software 4.1 Addendum 1 - February 2010.



Sequencing of PCR products generated with SYBR Green I can be performed, after purifying with the High Pure PCR Product Purification Kit, to remove the SYBR Green I dye.



Cloning of PCR products amplified with the LightCycler^® RNA Master SYBR Green I may be done by direct T/A cloning, blunt-end cloning, or after restriction enzyme digestion. It is not necessary to purify the PCR products prior to cloning. As host, use a UNG-negative E. coli strain, as the LightCycler^® Reagent Kits all contain dUTP.



SYBR Green I is not suitable for detecting single point mutations.
SYBR Green I is most likely only suitable for the screening of large deletion mutations, because the SYBR Green I detection format can only resolve a single mutation in a 50 bp fragment. Customer data suggest that it is possible to detect small deletions/insertions (10 to 20 bp) with SYBR Green I. In general, Roche recommends the format of the HybProbe probe for mutation analysis.", "Country": "XG", "Code": "Protocols - Help Corner", "Name": "Protocols - Help Corner" }, { "Language": "en", "Value": "

Vial/Bottle	Cap	Label	Function / Description	Content
1	red	LightCycler® RNA Master SYBR Green I, LC RNA Master SYBR Green l, 2.7x conc.	Contains Tth DNA Polymerase, reaction buffer, dNTP mix (with dUTP instead of dTTP), and SYBR Green I.	3 vials, 250 μl each
2	colorless	LightCycler® RNA Master SYBR Green I, Mn(OAc)2 stock solution, 50 mM	To adjust Mn(OAc)2 concentration in the reaction mix.	1 vial, 1 ml
3	colorless	LightCycler® RNA Master SYBR Green I, ​​​​​​​Water, PCR Grade	To adjust the final reaction volume.	2 vials, 1 ml each

", "Country": "XG", "Code": "Content", "Name": "Content" } ] } } ] }