For general laboratory use. Others FastStart Essential DNA Green Master GLU FastStart Essential DNA Green Master 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. 3.8.1.6.3.2 06924204001 FS Essential DNA Green Master,10x5 ml FastStart Essential DNA Green Master 04038377028729 Reagents, kits 1 kit 10 x 500 reactions of 20 μL final volume each false 06402712001 FastStart Essential DNA Green Master FastStart Essential DNA Green Master 04038377028095 Reagents, kits 1 kit 500 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 dye intercalates into the DNA helix. 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® PRO and LightCycler® 96 Instrument’s optics match 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® Systems are: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.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' light emission upon excitation.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.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 worksFastStart Essential DNA Green Master is a ready-to-use reaction mix designed specifically for applying the SYBR Green I detection format in the LightCycler® 480 Multiwell Plates, or LightCycler® 8-Tube Strips on the LightCycler® PRO or LightCycler® 96 Instrument. It is used to perform hot start PCR. Hot start PCR has been shown to significantly improve the specificity and sensitivity of PCR 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, 5 to 10 minutes) before cycling begins. Activation does not require the extra handling steps typical of other hot start techniques. en The FastStart Essential DNA Green Master is suited for hot start PCR applications. In combination with the LightCycler® PRO or LightCycler® 96 Systems and suitable PCR primers, this kit allows very sensitive detection and quantification of defined DNA sequences. The kit can also be used to perform two-step RT-PCR, and can be used with heat-labile Uracil-DNA Glycosylase to prevent carryover contamination during PCR.In principle, the kit can be used for the amplification and detection of any DNA or cDNA target. However, each amplification protocol will need to be adapted to the reaction conditions of the LightCycler® PRO or LightCycler® 96 Instruments, and specific PCR primers will need to be designed for each target. en FastStart Essential DNA Green Master is a ready-to-use reaction mix using the SYBR Green I detection format on the LightCycler® PRO and the LightCycler® 96 Instrument. It is used to for hot-start PCR in 8-Tube Strips or multiwell plates. Hot-start PCR significantly improves the specificity and sensitivity of PCR by minimizing the formation of nonspecific amplification products at the beginning of the reaction. en