PowerTrack SYBR Green Master Mix

PowerTrack SYBR Green Master Mix is a pre-formulated, optimized, universal 2X master mix for real-time PCR. It contains antibody-mediated Taq DNA polymerase with a hot-start mechanism that provides tight control over Taq enzyme activation and helps prevent undesirable early activity of the polymerase at low temperatures that can lead to nonspecific amplification. Building on over 20 years of innovation and product excellence in qPCR, PowerTrack SYBR Green Master Mix is designed for exceptional performance in your most challenging real-time PCR applications.

In an evaluation of 24 different primer sets used with PowerTrack SYBR Green Master Mix, a single melt curve was obtained in 100% of reactions. In contrast, nonspecific amplification was observed for some of the same targets with several master mixes from other suppliers, as shown by multiple peaks in the melt curves (Figure 1). Validation of primer specificity in SYBR Green reactions is essential to data quality and validity [1]. The high specificity enabled by PowerTrack SYBR Green Master Mix allows you to spend less time optimizing and redesigning primers to get high quality data.

Amplification curves were obtained for the PGK1 gene over a 5-log dilution series of UHR cDNA.  PowerTrack SYBR Green Master Mix delivers accurate results over a wide dynamic range of concentrations as shown by tight curves between replicates and superior PCR efficiency (Figure 2).

Figure 2.  Reactions were run in quadruplicate on the QuantStudio 5 Real-Time PCR System with the 384-well block according to protocol.  10-µL reactions were run in quadruplicate using 60ºC annealing primer Tm. PGK1 = high expressor; ARL1, IGF2, and TCF25 = medium expressor.

Reproducibility is another important measure of data quality in real-time PCR, and reproducibility often declines at low template concentrations, where the impacts of variability are exacerbated. However, PowerTrack SYBR Green Master Mix demonstrated excellent reproducibility over a wide dynamic range with a variety of targets and RT kits tested (Figure 3). Tighter reproducibility allows for greater statistical significance when analyzing low-abundance transcripts and smaller fold changes.

Figure 3. Reproducibility of data. Six assays (PGK1, ARL1, SNF8, DF, GAPDH, and Corf1 19) were run in quadruplicate with UHR cDNA generated from four different reverse transcription (RT) kits (SuperScript IV VILO, High Capacity RNA to cDNA, iScript cDNA synthesis, and Quantitect RT kits) run with 6-fold dilution series with 400 nM primer concentration on the QuantStudio 5 Flex Real-Time PCR System according to each master mix manufacturer’s recommended protocol.

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PowerTrack SYBR Green Master Mix can be used in either standard or fast cycling mode and is compatible with all Applied Biosystems real-time PCR instruments. It is also compatible with the Bio-Rad CFX96, CFX384, and iQ5; Roche LightCycler 480; and Agilent Mx3005P systems.  For optimal results, primer concentrations should be in the range of 300–800 nM.

ROX dye is an inert reference dye used in RT-qPCR that is included in our master mixes.  It is effective in normalizing fluorescence across all samples.  It removes fluorescence variations, such as those caused by bubbles, from the data.  Applied Biosystems master mixes contain a proprietary ROX dye specifically formulated for a wide range of real-time PCR instruments and compatible with a wide range of instrument light sources and filter sets.

Contamination in labs that routinely run PCR is a major concern for many researchers and is the source of most false positives. The inclusion of UNG and dUTP in the PowerTrack SYBR Green Master Mix allows any previously amplified PCR products to be degraded and helps prevent contamination of subsequent qPCR reactions. Utilization of a heat-labile UNG enzyme allows for effective contamination control.

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Reference

1. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009 55(4):611-22.