One universal master mix for all qPCR instruments
PowerUp™ SYBR™ Green Master Mix features include:
- Exceptional specificity with dual hot-start mechanism
- Tight reproducibility in Cts over a broad dynamic range
- Compatible with standard or fast cycling for results in <50 minutes
- Formulated with UNG/dUTP to prevent contamination of carryover PCR products
- Room temperature stable for 72 hours after plates are prepared for qPCR
- Broad instrument compatibility
PowerUp™ SYBR® Green Master Mix contains proprietary Dual-Lock™ Taq DNA Polymerase enzyme, which uses a combination of two hot-start mechanisms to control its activity. Providing tight control over Taq activation, this dual hot-start approach helps prevent undesirable early activity of the polymerase at low temperatures that can lead to non-specific amplification. Formulation of the Dual-Lock™ Taq into a convenient 2X pre-mix containing an optimized buffer system, proprietary additives, and detergents further improves the specificity of PowerUp™ SYBR® Green Master Mix.
In an evaluation of 24 different primer sets with PowerUp™ SYBR® Green Master Mix, a single melt curve was obtained in 100% of reactions without the need for primer optimization or redesign. In contrast, nonspecific amplification was seen for some of the same targets in several competitors’ mixes as shown by multiple peaks in the melt curve (Figure 1). Validation of specificity in SYBR® reactions is essential to data quality and validity (Bustin et al; 2009). The high specificity of the PowerUp™ SYBR® Green Master Mix allows you to spend less time and money optimizing and redesigning primers to get high-quality data.
Reproducibility is another important measure of data quality in real-time PCR reactions, and reproducibility often decays at low template concentration where the impacts of variability are exacerbated. However, PowerUp™ SYBR® Green Master Mix, with its dual hot-start Taq DNA polymerase, demonstrates excellent reproducibility over a wide dynamic range for a variety of targets tested (Figure 2). Tighter reproducibility allows for greater significance in analyzing low-abundant transcripts and smaller fold changes.
PowerUp™ SYBR® Green Master Mix provides robust results from 100 ng to 0.1 pg cDNA per reaction.1 Generating consistent amplification across a wide dynamic range is fundamental to qPCR methodology and use of the ΔΔCt method for gene expression analysis. Unlike competitor mixes that can exhibit inhibition at high cDNA inputs and low correlation coefficients over a dynamic range experiment, PowerUp™ SYBR® Green Master Mix efficiency is maintained over 6 logs of input to deliver maximum confidence in data quality (Figure 3, Table 1).
1Dynamic range is a property of both the assay and template concentration in the sample, as well as in the master mix, thus individual results may vary.
Figure 3: Robust performance of PowerUp™ SYBR® Green Master Mix over broad dynamic range. Amplification curves were obtained for the PGK1 gene over a 6 log dilution series of human universal reference cDNA. Reactions were run in triplicate on a QuantStudio® 7 instrument under Cycling conditions according to manufacturer's recommended protocol. Competitor mixes demonstrated greater inhibition as evidenced by the shape of the curves and the narrowed spacing between the dilution points at the highest cDNA inputs. In addition, several competitor mixes failed to reliably detect the lowest dilution point.
|PowerUp™ SYBR® Master Mix||5||5||4||6|
Table 1. Dynamic range comparison of competitor master mixes across 4 assays. The numbers indicate the linear range of 10-fold dilutions that can be measured by each mix for each target. For example, 6 points span seven 10-fold dilutions from 100 ng down to 0.1 pg of universal human reference cDNA per 10 μL reaction.
PowerUp™ 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.2 It is also compatible with the Bio-Rad IQ™5 and CFX systems, Roche LightCycler® 480, and Agilent Mx3005P® systems. For optimal results, recommended primer concentrations should be between 300–800 nM.
2 For optimal performance, recommended with standard cycling only for Applied Biosystems® 7900 systems.
Contamination in labs that routinely carry out PCR is a major concern for many researchers and is the source of most false positives. The inclusion of UNG and dUTP in the PowerUp™ SYBR® Green Master Mix degrades any previously amplified PCR products that might contaminate subsequent qPCR reactions. Use of a heat-labile UNG enzyme allows for effective contamination control within the PowerUp™ SYBR® Green Master Mix, without impacting downstream analyses of the PCR product, such as melt or gel analysis.
PowerUp™ SYBR® Green Master Mix, with its Dual-Lock™ Taq DNA Polymerase, has been shown to generate consistent performance in preassembled reactions for up to 72 hours (Table 2). The stability of this mix provides users the flexibility to set up plates prior to cycling without impacting results.3
3 Pre-PCR stability is influenced not only by the master mix, but by the target being analyzed as well. For maximum confidence, researchers should confirm stability profiles of their specific targets.
|Target||T-0 hr||T-72 hr||∆Ct|
Table 2. Pre-PCR stability. Several targets were amplified using PowerUp™ SYBR® Green Master Mix from 1 ng human cDNA on a StepOnePlus™ Real-Time PCR System either immediately following reaction setup or after 72 hours incubation at room temperature in the dark. Single melt curves were confirmed for these assays at both the 0 hr and 72 hr time points.
For Research Use Only. Not for use in diagnostic procedures.