LightCycler 480 SYBR Green I Master
LightCycler® 480 SYBR Green I Master is designed for research studies. When used with the LightCycler® 480 System, this kit is ideally suited for hot start PCR applications. In combination with the LightCycler® 480 System 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. It can also 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.
Benefits of the LightCycler 480 SYBR Green I Master
- Save time using this convenient, ready-to-use 2x concentrated hot start master mix.
- Minimize pipetting steps and contamination risk with this all-in-one mix.
- Perform sensitive, specific, and quantitative PCR or sequence detection using the LightCycler® 480 Instrument.
- Eliminate the time-consuming MgCl2 titration step.
- Achieve consistent, high-quality performance with the LightCycler® 480 Instrument and Multiwell Plates.
How the LightCycler 480 SYBR Green I Master works
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 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. 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:
- 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 to emit light 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.
Figure: Melting curve analysis of amplified samples with cDNA derived from 5 × 10 5 , 5 × 10 4 , 5 × 10 3 , 5 × 10 2 , and 50 copies of in vitro transcript as starting template. As a negative control, template DNA was replaced by Water, PCR grade.
