qPCR Mastermix Probe No Rox
qPCR Mastermix Probe No Rox is optimized for probe-based real-time PCRs and contains all necessary components for quantitative PCR except template DNA, primer and probe. qPCR Mastermix Probe No Rox is 5x concentrated and contains optimized components such as our Hot-Start Taq DNA Polymerase in a proprietary buffer system that allows the detection of "low-copy-number targets".
This kit does not contain the reference dye rox.
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
- No reference dye
Good to know
How does probe-based qPCR detection work?
In probe-based real-time PCR (e.g., using a TaqMan probe), detection is achieved using a sequence-specific fluorescent probe that binds to the target DNA in addition to the primers. The probe carries a reporter dye at the 5’ end and a quencher at the 3’ end. The quencher suppresses the fluorescence of the reporter as long as both are in close proximity to each other. During the elongation phase, the DNA polymerase degrades the bound probe due to its 5’→3’ exonuclease activity. This separates the reporter and quencher, and the reporter emits fluorescence (see figure). With each subsequent DNA amplification, the fluorescence increases proportionally. The signal is measured in real time and allows for the quantitative determination of the target DNA.
Which Taq DNA polymerase is used in the master mix?
The Hot-Start Taq DNA polymerase included is a further development of our thermostable Taq DNA polymerase (isolated from the thermophilic bacterium Thermus aquaticus). Due to an introduced modification, 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.