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This software provides precise estimations of the melting temperatures (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.", "Language": "en", "Country": "XG", "Code": "Background Information - Help Corner" }, { "Name": "Troubleshooting - Help Corner", "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^® FastStart DNA Master SYBR Green I away from light!", "Language": "en", "Country": "XG", "Code": "Troubleshooting - Help Corner" }, { "Name": "Assay Time", "Value": "

Procedure	Assay Time [min]
Optional: dilution of template DNA	5
PCR Setup	15
LightCycler® Carousel-Based System PCR run (including Melting Curve)	45
Total Assay Time	65

", "Language": "en", "Country": "XG", "Code": "Assay Time" }, { "Name": "Applications", "Value": "LightCycler^® FastStart DNA Master SYBR Green I is an easy-to-use hot start reaction mix for sensitive PCR applications in LightCycler^® Capillaries, using SYBR Green I as detection format. It is an ideal master mix for performing quantitative PCR, as well as SNP and mutation detection, and can also be used in two-step RT-PCR.
Note: LightCycler^® FastStart DNA Master SYBR Green I can be used in conjunction with heat-labile Uracil DNA Glycosylase for carryover prevention during PCR.", "Language": "en", "Country": "XG", "Code": "Applications" }, { "Name": "Principle", "Value": "Generation of PCR products can be detected by measurement of the SYBR Green I fluorescence signal. SYBR Green I intercalates into the DNA double helix. In solution, the unbound SYBR Green I dye exhibits very little fluorescence; however, fluorescence (wavelength, 530 nm) is greatly enhanced upon DNA binding. Therefore, during PCR, the increase in SYBR Green I fluorescence is directly proportional to the amount of double-stranded DNA generated.
As SYBR Green I dye is very stable (only 6% of the activity is lost during 30 amplification cycles) and the LightCycler^® Instruments' optical filter set matches the wavelengths of excitation and emission, it is the reagent of choice when measuring total DNA.

The basic steps of DNA detection by SYBR Green I, during real-time PCR on the LightCycler^® Carousel-Based System are:

1. At the beginning of amplification, the reaction mixture contains the denatured DNA, the primers, and the SYBR Green I dye. The unbound SYBR Green I dye molecules weakly fluoresce, producing a minimal background fluorescence signal, which is subtracted during computer analysis.
2. After annealing of the primers, a few SYBR Green I dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green I dye molecules to emit light upon excitation.
3. During elongation, more and more SYBR Green I dye molecules bind to the newly synthesized DNA. If the reaction is monitored continuously, an increase in fluorescence is viewed in real time. Upon denaturation of the DNA for the next heating cycle, the SYBR Green I dye molecules are released and the fluorescence signal falls.
4. Fluorescence measurement at the end of the elongation step of every PCR cycle is performed to monitor the increasing amount of amplified DNA.

To prove that only your desired PCR product has been amplified, you may perform a Melting Curve analysis after PCR. 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 (Tm) of a particular DNA product (where the DNA is 50% double-stranded and 50% single-stranded). The most important factors that determine the Tm of dsDNA are the length and the GC-content of that fragment. 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. Checking the Tm of a PCR product can thus be compared with analyzing a PCR product by length in gel electrophoresis.LightCycler^® FastStart DNA Master SYBR Green I is a ready-to-use PCR reaction mix, designed specifically for real-time PCR assays using the SYBR Green I detection format on the LightCycler^® Carousel-Based System. It is used to perform hot start PCR in 20 μl glass capillaries. 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. et al., 1996), by minimizing the formation of nonspecific amplification products at the beginning of the reaction.
FastStart Taq DNA Polymerase is a chemically modified form of thermostable recombinant Taq DNA polymerase, that shows no activity up to +75°C. The enzyme is active only at high temperatures, where primers no longer bind nonspecifically. The enzyme is completely activated (by removal of blocking groups) in a single pre-incubation step (+95°C, 10 minutes) before cycling begins. Activation does not require the extra handling steps typical of other hot start techniques.
LightCycler^® FastStart DNA Master SYBR Green I provides convenience, excellent performance, and reproducibility, as well as minimizing contamination risk. All you need to supply is template DNA, PCR primers, and additional MgCl₂ (if necessary).

In principle, the LightCycler^® FastStart DNA Master SYBR Green I can be used for the amplification and detection of any DNA or cDNA target. However, you would need to optimize the detection protocol to the reaction conditions of the LightCycler^® Carousel-Based System and design specific PCR primers for each target. Refer to the LightCycler^® Operator’s Manual for general recommendations.

", "Language": "en", "Country": "XG", "Code": "Principle" }, { "Name": "Content", "Value": "

Vial / Bottle	Cap	Label	Function / Description	Catalog Number	Content
1a	colorless	LightCycler® FastStart DNA Master SYBR Green I, LC FastStart Enzyme	Ready-to-use hot start PCR reaction mix (after pipetting 10 μl from Vial 1a into one Vial 1b). Contains FastStart Taq DNA Polymerase, reaction buffer, dNTP mix (with dUTP instead of dTTP), SYBR Green I dye, and 10 mM MgCl2.	03 003 230 001	1 vial 1a, 3 vials 1b, for 3 × 64 μl each LightCycler® FastStart DNA Master SYBR Green I, 10x conc.
1b	green	LightCycler® FastStart DNA Master SYBR Green I, LC FastStart Reaction Mix SYBR Green I, 10x conc.	12 239 264 001	5 vials 1a, 15 vials 1b, for 15 × 64 μl each LightCycler® FastStart DNA Master SYBR Green I, 10x conc.
2	blue	LightCycler® FastStart DNA Master SYBR Green I, MgCl2 stock solution, 25 mM	To adjust MgCl2 concentration in the reaction mix.	03 003 230 001	1 vial, 1 ml
12 239 264 001	2 vials, 1 ml each
3	colorless	LightCycler® FastStart DNA Master SYBR Green I, Water, PCR Grade	To adjust the final reaction volume.	03 003 230 001	2 vials, 1 ml each
12 239 264 001	7 vials, 1 ml each

", "Language": "en", "Country": "XG", "Code": "Content" }, { "Name": "Protocols - Help Corner", "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.


To perform Touchdown PCR with LightCycler^® FastStart DNA Master SYBR Green I, decrease the annealing temperature during the first 20 cycles by 10°C in steps of 2°C: start from an initial temperature +6 to +8°C above the melting temperature of your PCR primers down to a temperature 2°C above the final annealing temperature. Use a Ramp Rate of 0.5°C per cycle. The final 25 cycles are then run at the final annealing temperature.

An example touchdown protocol for a PCR primer set of Tm 56°C might be as follows:
95°C, 10 min
95°C, 10 s / 62°C 5 s / 72°C 10 s (x 4 cycles)
95°C, 10 s / 60°C 5 s / 72°C 10 s (x 4 cycles)
95°C, 10 s / 58°C 5 s / 72°C 10 s (x 4 cycles)
95°C, 10 s / 56°C 5 s / 72°C 10 s (x 4 cycles)
95°C, 10 s / 54°C 5 s / 72°C 10 s (x 4 cycles)
95°C, 10 s / 52°C 5 s / 72°C 10 s (x 25 cycles)
Note that elongation time depends on product length, follow the general rule: elongation time (sec) = amplicon length [bp]/25.


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^® FastStart DNA 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.


LightCycler^® FastStart DNA Master SYBR Green I can be used for the study of hypermethylation. Please refer to the following articles:

1. Florl, AR. et al. (2005). Screening for DNA Hypermethylation Using the LightCycler Instrument. Biochemica 1, 4-6.
2. Thomassin, H. et al. (2004). MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome. Nucleic Acids Res. 32, e168.

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.", "Language": "en", "Country": "XG", "Code": "Protocols - Help Corner" }, { "Name": "Product Description", "Value": "Easy-to-use hot start reaction mix for PCR, using SYBR Green I with the LightCycler^® Carousel-Based System.", "Language": "en", "Country": "XG", "Code": "Product Description" }, { "Name": "Additional Equipment and Reagent Required", "Value": "

• Nuclease-free, aerosol-resistant pipette tips
• Pipettes with disposable, positive-displacement 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 (optional)

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

", "Language": "en", "Country": "XG", "Code": "Additional Equipment and Reagent Required" } ] } } ] }