qPCR Mastermix EvaGreen® Rox
qPCR Mastermix EvaGreen® Rox is optimized for EvaGreen®-based real-time PCRs and contains all necessary components for quantitative PCR except template DNA and primers. The 5x concentrated kit is highly specific and provides reproducible results for low copy DNA targets, long template or GC-rich DNA targets.
This kit contains the reference dye Rox. Rox is a passive, internal reference dye used to normalize the fluorescence reporter signal during qPCR.
Properties
- 5x concentrated, ready-to-use master mix
- Thermostable hot-start Taq DNA polymerase (Stable for up to one month at room temperature!)
- 12.5 mM MgCl₂ (= 2.5 mM in 1x solution)
- Nucleotides (dATP, dGTP, dCTP, dTTP) in an optimized composition
- EvaGreen® dye
- ROX dye
Good to know
How does real-time detection with EvaGreen® work?
In qPCRs (quantitative polymerase chain reactions) using the dye EvaGreen®, detection is based on the dye’s binding to double-stranded DNA. EvaGreen® only fluoresces when it binds to double-stranded DNA. At the start (denaturation phase) of the qPCR, the DNA is single-stranded, which is why no (or hardly any) fluorescence is detectable. When the DNA is amplified (elongation phase), double-stranded DNA is formed again, to which EvaGreen binds. The fluorescence signal increases (see figure) and is measured in real time.
Why EvaGreen®?
The EvaGreen® dye is characterized by its high stability and good signal intensity. It has a fluorescence spectrum very similar to that of SYBR® Green I, but exhibits lower PCR inhibition than SYBR® Green I. EvaGreen® is neither mutagenic nor cytotoxic and is compatible with all common real-time PCR cyclers—simply select the default setting for SYBR® Green or FAM!
Which Taq DNA polymerase is used in the master mix?
The included Hot-Start Taq DNA polymerase is a further development of our thermostable Taq DNA polymerase (isolated from the thermophilic bacterium Thermus aquaticus). Thanks to a modification introduced, the enzymatic activity of our Hot-Start Taq DNA polymerase is only released after a single incubation at 95°C for 15 minutes.
Our Hot-Start Taq DNA polymerase is a highly processive 5'-3' DNA polymerase with 5'-3' exonuclease activity. It completely lacks 3'-5' exonuclease activity. Additionally, the enzyme adds single nucleotides (almost exclusively adenosine) to the 3’ ends of the DNAs, enabling TA cloning without further modifications.
With which instruments is the Mastermix compatible?
The Mastermix is compatible with all standard plate-based real-time cyclers, regardless of Rox requirements.
It is suitable for all “Standard Cycling Mode,” “Standard,” and “Fast” real-time cyclers.
EvaGreen® is compatible with all common real-time PCR cyclers—simply select the default setting for SYBR® Green or FAM!