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In principle, the kit can be used for the amplification and detection of any DNA or cDNA target.", "Language": "en", "Country": "XG", "Code": "Applications" }, { "Name": "Principle", "Value": "LightCycler^® 480 SYBR Green I Master is a ready-to-use reaction mix designed specifically for applying the SYBR Green I detection format on the LightCycler^® 480 Instrument. It is used to perform hot start PCR in 96- or 384-multiwell plates. Hot start PCR has been shown to significantly improve the specificity and sensitivity of PCR [Chou Q, et al, 1992 and Kellogg DE, et al, 1994] 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 non-specifically. The enzyme is completely activated (by removal of blocking groups) in a single pre-incubation step (95°C, 5 - 10 minutes) before cycling begins. Activation does not require the extra handling steps typical of other hot start techniques.

Generation of PCR products can be detected by measurement of the SYBR Green I fluorescence signal. SYBR Green I intercalates into the DNA helix (5). In solution, the unbound 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. Since SYBR Green I dye is very stable (only 6% of the activity is lost during 30 amplification cycles) and the LightCycler^® 480 Instrument's 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^® 480 System are:
 

1. At the beginning of amplification, the reaction mixture contains the denatured DNA, the primers, and the dye. The unbound dye molecules weakly fluoresce, producing a minimal background fluorescence signal which is subtracted during computer analysis.
2. After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green I molecules to emit light upon excitation.
3. During elongation, more and more 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 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 97°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 non-specific 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.", "Language": "en", "Country": "XG", "Code": "Principle" }, { "Name": "Product Description", "Value": "The LightCycler^® 480 SYBR Green I Master is a one-component hot start reaction mix for PCR. It contains FastStart Taq DNA Polymerase and DNA double-strand-specific SYBR Green I dye for PCR product detection and characterization. Since the mix is provided as an easy-to-use all-in-one master 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 DNA or cDNA), and is suited for high-throughput applications in 96- or 384-well plates on the LightCycler^® 480 Instrument.", "Language": "en", "Country": "XG", "Code": "Product Description" }, { "Name": "Content", "Value": "

Vial / Bottle	Cap	Label	Function	Catalog Number	Content
1	green	LightCycler® 480 SYBR Green I Master, 2x conc.	Ready-to-use hot start PCR mix Contains FastStart Taq DNA Polymerase, reaction buffer, dNTP mix (with dUTP instead of dTTP), SYBR Green I dye, and MgCl2	04 707 516 001	5 vials, 1 ml each
04 887 352 001	10 vials, 5 ml each
2	colorless	Water, PCR-grade	To adjust the final reaction volume	04 707 516 001	5 vials, 1 ml each
04 887 352 001	2 vials, 25 ml each

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