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"ProductSpec": [ { "ProductSpecVariant": { "Chapters": [ { "Language": "en", "Value": "Store the kit at −15 to −25°C.", "Country": "XG", "Code": "Storage Conditions (Product)", "Name": "Storage Conditions (Product)" }, { "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 Amplification Kit SYBR Green I away from light!", "Country": "XG", "Code": "Troubleshooting - Help Corner", "Name": "Troubleshooting - 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": "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 Amplification Kit 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": "Generation of PCR products can be detected by measurement of the SYBR Green I fluorescence signal. SYBR Green I dye (already included in the Reaction Mix of the LightCycler^® RNA Amplification Kit SYBR Green I) will emit a fluorescence signal (530 nm) only when intercalated into the DNA double helix. Therefore during PCR, the increase in SYBR Green I fluorescence is directly proportional to the amount of double-stranded DNA generated.
Specificity and sensitivity of amplification reactions detected with SYBR Green I dye is greatly enhanced by combining amplification with a Melting Curve analysis. In Melting Curve analysis, the reaction mixture is slowly heated to 95°C, which causes melting of double-stranded DNA and a corresponding decrease of SYBR Green I fluorescence. The instrument continuously monitors this fluorescence decrease and displays it as melting peaks. Each melting peak represents the characteristic melting temperature of a particular DNA product (where the DNA is 50% double-stranded and 50% single-stranded). If PCR generated only one amplicon, Melting Curve analysis will show only one melting peak. If primer-dimers or other nonspecific products are present, they will be shown as additional melting peaks.The LightCycler^® RNA Amplification Kit SYBR Green I is designed specifically for the SYBR Green I detection format using the LightCycler^® Carousel-Based System. It is used to perform one-step RT-PCR in 20 μl glass capillaries. Amplification and online monitoring of the template RNA is achieved by a combined procedure on the LightCycler^® Carousel-Based System Instruments. The results are interpreted directly after completing the PCR. The amplicon is detected by measurement of the SYBR Green I fluorescence signal.
The LightCycler^® RNA Amplification Kit SYBR Green I provides convenience, high performance, reproducibility, and minimizes contamination risk. All you need to supply is template RNA, PCR primers, and additional MgCl₂ (if necessary).

In principle, the LightCycler^® RNA Amplification Kit SYBR Green I can be used for the amplification and detection of every RNA target. However, you would need to optimize each amplification protocol to the reaction conditions of the LightCycler^® Carousel-Based System.", "Country": "XG", "Code": "Principle", "Name": "Principle" }, { "Language": "en", "Value": "

Procedure	Assay Time [min]
PCR Setup	15
Reverse Transcription	10
LightCycler®Carousel-Based System PCR run	25
Total Assay Time	50

", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "

Vial/Bottle	Cap	Label	Function/Description	Content
1	green	LightCycler® RNA Amplification Kit SYBR Green I, LC RT-PCR Enzyme Mix	Enzyme mix for RT-PCR.	2 vials, 20 μl each
2	green	LightCycler® RNA Amplification Kit SYBR Green I, LC RT-PCR Reaction Mix, SYBR Green I, 5x conc.	Reaction mix for RT-PCR. Contains reaction buffer, dNTP mix (with dUTP instead of dTTP), SYBR Green I dye, and 15 mM MgCl2.	3 vials, 128 μl each
3	blue	LightCycler® RNA Amplification Kit SYBR Green I, MgCl2 stock solution, 25 mM	To adjust MgCl2 concentration.	1 vial, 1 ml
4	colorless	LightCycler® RNA Amplification Kit SYBR Green I, Water, PCR Grade	To adjust the final reaction volume.	2 vials, 1 ml each
5	colorless	LightCycler® RNA Amplification Kit SYBR Green I, Resolution solution, 5x conc.	To amplify RNA templates of high GC-content or high degree of secondary structures.	1 vial, 1 ml

", "Country": "XG", "Code": "Content", "Name": "Content" }, { "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": "The LightCycler^® RNA Amplification Kit SYBR Green I is designed for use in life science research. The kit provides reagents, including RT-PCR enzyme mix, reaction mix, MgCl₂, Resolution Solution, and Water, PCR Grade for one-step RT-PCR in glass capillaries using the LightCycler^® Carousel-Based System and SYBR Green I as detection format.
In addition, the kit can be used with Uracil-DNA Glycosylase, heat labile to prevent carryover contamination during PCR.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "Easy-to-use Reaction Mix for One-Step RT-PCR, using SYBR Green I with the LightCycler^® Carousel-Based System.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" } ] } } ] }