For life science research only. Not for use in diagnostic procedures. Others LightCycler FastStart DNA Master SYBR Green I LSR LightCycler® FastStart DNA Master SYBR Green I 67 1280837236338 SYBR Green I is manufactured by Molecular Probes, Inc., and is provided under license from Molecular Probes, Inc., for direct research use for PCR, where the dye is present during the PCR. 3.5.5.1.3.1 03003230001 LC FS DNA Master SYBR Green I, 96 react. LightCycler FastStart DNA Master SYBR Green I 04038377016870 Reagents, kits 1 kit 96 reactions of 20 μl final volume each false 12239264001 LC FS DNA Master SYBR Green I,480 react. LightCycler FastStart DNA Master SYBR Green I 04038377016856 Reagents, kits 1 kit 480 reactions of 20 μl final volume each false 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: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.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.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.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.How this Product WorksLightCycler® 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.The amplicon size should not exceed 1 kb in length. For optimal results, select a product length of 700 bp or less. en Easy-to-use hot start reaction mix for PCR, using SYBR Green I with the LightCycler® Carousel-Based System. en LightCycler® FastStart DNA Master SYBR Green I is ideally suited for hot start PCR applications. In combination with the LightCycler® Carousel-Based System and suitable PCR primers, this kit enables very sensitive detection and quantification of defined DNA sequences. Furthermore, the kit can be used to perform two-step RT-PCR, in combination with a reverse transcription kit for cDNA synthesis.LightCycler® FastStart DNA Master SYBR Green I can also be used with LightCycler® Uracil-DNA Glycosylase to prevent carryover contamination during PCR. en