Processing
Since its beginnings, IDT has worked to improve sustainability practices. Much of the focus has been on manufacturing processes to reduce air emissions, water and energy usage, and hazardous and landfill waste production. Here, we show results from experiments supporting ambient shipping for PrimeTime® Gene Expression Master Mix to further mitigate environmental impacts of our products. Eliminating shipping in insulated containers with gel packs and dry ice reduces shipping costs, saving you money. It also lessens detrimental environmental impacts from manufacturing insulated foam containers, addition of dry ice or gel packs, and waste disposal of these items in landfills or incinerators.
PrimeTime Gene Expression Master Mix for probe-based qPCR assays
For consistency and convenience, many researchers use commercial master mixes that have been optimized, often with enhancers and stabilizers, to work in most standard assays. IDT scientists have developed a robust qPCR master mix for probe-based assays for qPCR. The versatile PrimeTime Gene Expression Master Mix produces consistent results under both standard and fast cycling conditions, and is compatible with a wide range of real-time PCR instruments.
With excellent benchtop stability, PrimeTime Gene Expression Master Mix is compatible with overnight and high throughput experiments. Further extending these thermal stability tests, our research group has demonstrated that master mix function is not affected by conditions that could be encountered during ambient shipping. We continue to observe consistent qPCR performance after heat-stressing the master mix at 50°C for up to 7 days (Figure 1), and after 20 freeze-thaw cycles ]. The master mix retains its ability to provide high efficiency PCR results, despite exposure to temperature conditions that are more extreme than those encountered during routine ambient shipping or laboratory use.
Figure 1. Obtain consistent, high PCR efficiency and no change in Cq values, even after PrimeTime Gene Expression Master Mix is heated to 50°C for up to 7 days. PrimeTime master mix was incubated at 50°C for 1, 3, 5, or 7 days, while a control master mix sample was stored frozen (–20°C) until use. The graph shows representative amplification curves from qPCRs that included the heat-treated or control master mix with PrimeTime qPCR HPRT Assays (Assay ID: Hs.PT.58v.45621572). The reactions remained at room temperature for 24 hr before running the thermal cycler: control master mix (gray) and the 1-, 3-, 5-, and 7-day, heat-treated master mix (green, light blue, dark blue, and orange, respectively).
Visit www.idtdna.com/qPCRmastermixfor more information about the PrimeTime Gene Expression Master Mix. To see additional data from our thermal stability studies, download the white paper, Ambient shipping of PrimeTime Gene Expression Master Mix.
What master mixes do
Table 1 provides a brief description of the role of the main components of a master mix. Note that small changes in master mix formulation can significantly affect assay performance. Also, some formulations are designed for use with specific cycling conditions (e.g., standard or fast cycling conditions).
| Master mix component | Overview of function |
|---|---|
| Polymerase |
Master mixes normally contain modified, thermostable DNA polymerases to eliminate nonspecific priming that may occur before the initial denaturation step, which can skew Cq values. Hot-start polymerases, like the one used in PrimeTime Gene Expression Master Mix, are designed to be inactive at low temperatures. They are modified (with a blocking antibody, chemical modification, or aptamer) and require activation by heating (95°C for 2−10 min) during the initial denaturation step. |
| MgCL2 |
MgCl2 can affect the specificity of the qPCR. It stabilizes primer and probe interactions with DNA and is a cofactor for thermostable DNA polymerases. Typical final MgCl2 concentrations in qPCR can range from 1–6 mM. If MgCl2 levels are too low, polymerase conformation is suboptimal and PCR product yields will be low. If MgCl2 levels are too high, nonspecific amplification and misincorporation of dNTPs occurs. |
| dNTPs |
dNTPs are the building blocks for DNA synthesis.Typical reactions contain a final concentration of 10–200 µM of each dNTP. The optimal amount depends on the number of assays in the reaction (e.g., singleplex or multiplex), the size of the amplicon(s), and the concentration of MgCl2. Lower dNTP concentrations result in increased amplification fidelity, but lower yields, compared to higher dNTP concentrations. |
| Reference dye |
Some thermal cyclers require the use of an internal reference dye, such as ROX or a fluorescein dye, for fluorescent signal normalization across wells and to account for pipetting errors. Therefore, certain master mixes are available with different reference dye formulations. Refer to your instrument user manual for instructions. |
| Buffer |
Buffers are required to maintain optimum pH and salt conditions. |
| Additives |
Potential additives include denaturants or proteins that lower Tm or neutralize PCR contaminants from samples to improve specificity or PCR efficiency. |
