gene_expression

PrimeTime® Gene Expression Master Mix

Achieve quality performance under various experimental conditions with this thermostable probe-based qPCR master mix

PrimeTime Gene Expression Master Mix is a 2X solution containing a hot-start antibody, Taq polymerase, and other components needed for probe-based qPCR in two-step RT-PCR experiments. Reference dye is included separately.

  • Achieve high efficiency qPCR with fast or standard cycling
  • Use one mix for singleplex or multiplex reactions
  • Rely on exceptional benchtop stability to obtain consistent results from overnight experiments
  • Explore license-free options for commercial or diagnostic use

Ordering

Need 500 mL or more? Contact us for discounted pricing.

PrimeTime Gene Expression Master Mix is optimized to support probe-based qPCR assays for gene expression analysis. This master mix is guaranteed to provide assay efficiencies >90% when used with PrimeTime qPCR Assays in two-step RT-qPCR. It is also compatible with other primers and probes.

Each order includes a 2X master mix solution (antibody-mediated, hot-start DNA polymerase; dNTPs; MgCl2; enhancers; and stabilizers) and a separate reference dye stock solution.

PrimeTime Gene Expression Master Mix is shipped at ambient temperature. Elimination of shipping on dry ice saves your research money, minimizes shipping delays, and benefits the environment. To view our extensive testing that shows ambient shipping does not impact the function of the master mix, see our white paper.

For a general estimate, the following table provides the number of reactions (for 20 µL reaction volumes) for each order size:

ProductCatalog #Unit sizeReactions
PrimeTime Gene Expression Master Mix, 1 mL10557701 x 1 mL100 x 20 µL
PrimeTime Gene Expression Master Mix, 5 mL10557721 x 5 mL500 x 20 µL
PrimeTime Gene Expression Master Mix, 25 mL10557715 x 5 mL2500 x 20 µL

Overview of 5′ nuclease assays

Step 1—The primers and probe hybridize in a sequence-dependent manner to the complementary DNA strand. Because the probe is intact, the fluorophore and quencher are in close proximity and the quencher absorbs fluorescence emitted by the fluorophore.

Step 2—The polymerase extends from the primers and begins DNA synthesis.

Step 3—The polymerase reaches the probe and the exonuclease activity of the polymerase cleaves the hybridized probe. As a result of cleavage, the fluorophore is separated from the quencher and fluoresces.

Step 4—The fluorescence is detected by the real time instrument.

These steps are repeated for each PCR cycle and allow detection of specific products. When using intercalating dyes, such as SYBR® Green I, primer-dimers and non-specific products will also contribute to fluorescence. In contrast, the 5′ nuclease assay is specific and fluorescence will only be detected for the DNA sequence to which the probe and primers hybridize.

As good or better than other commercial master mixes

PrimeTime Gene Expression Master Mix delivers consistent results that are as good as or better than other commercially available master mixes tested. Cq values using PrimeTime master mix occurred approximately 2–4 cycles earlier in this representative experiment (Figure 1).

Figure 1. PrimeTime Gene Expression Master Mix delivers consistent results that are as good or better than 7 other commercially available master mixes tested. PCRs contained PrimeTime HPRT qPCR Assays, PrimeTime Gene Expression Master Mix with reference dye, and either cDNA or gBlocks® Gene Fragments as template. PCRs were run in triplicate on a 7900HT Real-Time PCR System (Thermo Fisher Scientific).

Compatible with PrimeTime qPCR Assays (and assays from other suppliers)

PrimeTime Gene Expression Master Mix delivers reliable results with singleplex and duplex amplification using qPCR assays from IDT or other suppliers. All assays exhibited 90–110% PCR efficiency with R2 >0.99 (Figures 2 and 3).

Figure 2. Reproducible results from singleplex (black) and duplex (red) qPCR using PrimeTime qPCR Assays. PCRs, containing PrimeTime HPRT and/or GUSB qPCR Assays, PrimeTime Gene Expression Master Mix with reference dye, and gBlocks® Gene Fragments (101–107copies) as template, were run on a 7900HT Real-Time PCR System (Thermo Fisher Scientific). HPRT Assay ID: Hs.PT.58v.45621572; GUSB Assay ID: Hs.PT.58v.27737538.

Figure 3. Reproducible results from singleplex (black) and duplex (red) qPCR using inventoried HPRT and GUSB qPCR Assays (Supplier A). PCRs, containing inventoried HPRT and/or GUSB qPCR assays from Supplier A, PrimeTime Gene Expression Master Mix with reference dye, and cDNA (0.0032–50 ng) as template, were run on a 7900HT Real-Time PCR System (Thermo Fisher Scientific).

Standard or fast cycling conditions

PrimeTime Gene Expression Master Mix performs well under standard or fast cycling conditions (Figures 4 and 5) on a variety of real-time qPCR instruments, including the 7900HT Real-Time (Thermo Fisher Scientific), QuantStudio™ 7 Flex (Thermo Fisher Scientific), CFX384 (BioRad), and LightCycler® 480 (Roche) qPCR systems.

Figure 4. High PCR efficiency under standard or fast cycling conditions. PCRs consisting of PrimeTime qPCR Assays, PrimeTime Gene Expression Master Mix, reference dye, and template were run on a 7900HT Real-Time PCR System (Thermo Fisher Scientific) using standard or fast cycling conditions. Standard cycling conditions: 3 min. 95°C; 49 x (15 sec. 95°C; 1 min. 60°). Fast cycling conditions: 3 min. 95°C; 49 x (5 sec. 95°C; 30 sec. 60°). This histogram shows the calculated PCR efficiency of 13 assays run under standard (orange) or fast (blue) cycling conditions using either diluted cDNA (0.016–50 ng) or gBlocks Gene Fragments (101–107 copies) as template. All assays exhibited 90–110% PCR efficiency with R2 >0.99.

Figure 5. Consistent Cq values under standard or fast cycling conditions. PCRs consisting of PrimeTime qPCR Assays, PrimeTime Gene Expression Master Mix, reference dye, and dilutions of cDNA (0.016–50 ng) were run on a 7900HT Real-Time PCR System (Thermo Fisher Scientific) using standard or fast cycling conditions. Standard cycling conditions: 3 min. 95°C; 49 x (15 sec. 95°C; 1 min. 60°). Fast cycling conditions: 3 min. 95°C; 49 x (5 sec. 95°C; 30 sec. 60°). At each concentration of cDNA, the difference in Cq values determined using standard or fast cycling conditions was <1.

Lot-to-lot consistency (0–24 hr at room temperature)

You can expect consistent performance, even when using different lots of PrimeTime Gene Expression Master Mix. This master mix has also shown temperature stability after 24 hours at room temperature, making it ideal for high-throughput applications or overnight experiments.

Figure 6. Dependable results across different batches of PrimeTime Gene Expression Master Mix, even after 24 hours at room temperature. PCR mixes consisting of PrimeTime qPCR Assays, PrimeTime Gene Expression Master Mix, reference dye, and varying amounts of gBlocks Gene Fragments (8 replicates) were assembled. A JANUS® automated, liquid-handling workstation (PerkinElmer) was used to load the reactions into PCR plates, which were run immediately (Day 0) or after 24 hours (Day 1) on a 7900HT Real-Time PCR System (Thermo Fisher Scientific). When comparing Day 1 and Day 0 results, the ∆Cq values for template levels >10 copies were <0.5. (A) Results using the PrimeTime HPRT qPCR Assay. (B) Results using the PrimeTime GUSB qPCR Assay.

Temperature stability

In addition to consistent performance after extended periods at room temperature, PrimeTime Gene Expression Master Mix has shown exceptional temperature stability, with no loss of amplification efficiency or degradation of components, even after a temperature stress tests (4 or 8 hours at 55°C, data not shown; up to 7 days at 50°C, Figure 7; up to 20 freeze-thaw cycles, Figure 8). For additional stability data, see the white paper.

Figure 7. Consistent performance after PrimeTime Gene Expression Master Mix was heated at 55°C for 4 or 8 hr. PrimeTime Master Mix was not heated or heated at 55°C (4 or 8 hr) before use in PCR with a PrimeTime qPCR Assay, reference dye, and varying amounts of cDNA (0.08–50 ng). An overlay of the amplification plots for the PCRs using not heated and heated master mix shows no effect on the PCR amplification curves. (A) Results using the PrimeTime HPRT qPCR Assay. (B) Results using the PrimeTime GUSB qPCR Assay.

Bulk orders

For orders >500 mL, contact us.

GMO or OEM

To inquire about license-free options for commercial or diagnostic use, contact Technical and Customer Support.