SYBR® Green dye is a fluorescent double-stranded DNA (dsDNA)- binding dye that is used to track the progress of DNA amplification in real-time PCR experiments. As the PCR reaction proceeds, at each round of amplification SYBR® Green dye binds to dsDNA as it polymerizes, resulting in an increase in the level of fluorescence at the end of each extension step. The quantity of dsDNA product in the reaction is proportional to the amount of fluorescence. SYBR® Green is not PCR inhibitory and provides accurate, reproducible results with high sensitivity over a broad dynamic range, and gives consistent results across different platforms. One advantage of using a SYBR® Green master mix is that it is cost effective, particularly when compared to probe-based detection systems. Generally, probes designed for PCR are oligos that have a fluorescent molecule on one end and a quencher on the other. When the oligo primes a PCR reaction and is incorporated into a PCR product, the fluorescent molecule is no longer quenched. Not only is SYBR® Green generally less expensive than using probes, it can be used with any combination of primers and template to monitor synthesis of all dsDNA sequences. Most probe systems require different probes for different sequences. The lack of a requirement for sequence- specific probes can result in shorter experimental preparation times.
SYBR® Green master mixes are compatible with any real-time PCR amplification protocol. SYBR® Green master mix applications include:
SYBR® Green master mixes are 2x concentrated, and contain dNTPs, MgCl2, and DNA polymerase.
SYBR® Green master mixes are available with other dyes including ROX. Many PCR machines require a passive reference dye. An internal reference dye, such as ROX, corrects well-to-well optical variations, and is used for fluorescent signal normalization.