PCR and qPCR
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A qPCR master mix stable enough for ambient shipping

Save money and protect the environment

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 (for more information, see the sidebar, What master mixes do). 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 [1]. 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 [1]. 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.

Visit www.idtdna.com/qPCRmastermix for more information about the PrimeTime Gene Expression Master Mix. To see additional data from our thermal stability studies, view or download the white paper, Exceptional thermal stability of PrimeTime® Gene Expression Master Mix enables ambient shipping [1].

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 to 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.

dNTPs

dNTPs are the building blocks for DNA synthesis.

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.

Table 1. Overview of PCR master mix components.


Overview of PCR master mix components

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).


Reference

  1. Thomas DM, Young M, et al. (2016) Strong thermal stability of the PrimeTime® Gene Expression Master Mix permits ambient shipping, saving laboratory and environmental resources. [Online] Coralville, Integrated DNA Technologies. White paper available at www.idtdna.com/pages/docs/default-source/qPCR/pcr-10032-an_1074903-ambientship-qpcrmastermix.pdf. [Accessed 1 Jun, 2016].


Product focus: Everything but your sample

All the reagents you need for successful qPCR assays are now available through IDT.

Additional resources

Application guide:

PrimeTime® qPCR application guide, 4th ed—Download this useful resource that provides experimental overviews, protocols, data analysis, and troubleshooting chapters.

Articles:

Design efficient PCR and qPCR primers and probes using online tools—To facilitate experimental planning, IDT offers a variety of free, online tools for oligonucleotide analysis and PCR primer design. This article provides an overview of our predesigned qPCR assays and the basics of designing customized PCR primers and hydrolysis probes with the PrimerQuest® Tool.

DECODED 4.2—special qPCR issue—Get this compendium of our newsletter articles about qPCR, all in one convenient volume.

Webinars:

High throughput qPCR: tips for analysis across multiple plates—Discover strategies for optimizing high throughput qPCR for expression profiling from prominent qPCR expert, Dr Mikael Kubista (TATAA Biocenter, Sweden). Download the slides here.

Troubleshooting qPCR — What Are My Amplification Curves Telling Me?—Learn about a variety of problematic qPCR issues and how they affect the appearance of the amplification curve. Download the slides here.

Author: Maureen Young, PhD, is a senior scientific writer at IDT.

© 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


Predesigned qPCR Assays

Probe-based qPCR assays for quantification of human, mouse, and rat gene expression. Order in plates or tubes.

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