Probe-Based qPCR Master Mix

PrimeTime® Gene Expression Master Mix is a 2X solution designed for use in two-step RT-qPCR.

  • Achieve high efficiency qPCR under fast or standard cycling conditions
  • Multiplex without loss of sensitivity
  • Obtain consistent results from overnight experiments by capitalizing on exceptional benchtop stability

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.

As part of our sustainability efforts, IDT scientists have conducted extensive testing to show that ambient shipping conditions do not impact the function of the master mix. Elimination of shipping on dry ice saves your research money, minimizes shipping delays, and benefits the environment. View the data in the ambient shipping white paper.

Product Catalog #Unit Size Reactions
1 mL PrimeTime® Gene Expression Master Mix10557701 x 1 mL100 x 20 µL
5 mL PrimeTime® Gene Expression Master Mix10557721 x 5 mL500 x 20 µL
25 mL PrimeTime® Gene Expression Master Mix10557715 x 5 mL2500 x 20 µL

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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: Nucleotide extension is completed, and the emitted fluorescence is detected by the real-time instrument.

These steps are repeated for each PCR cycle and allow detection of specific products, because fluorescence is only detected for the DNA sequence to which the probe and primers hybridize.

Overview of probe-based 5′ nuclease assays

Protocol


White paper


Application guide


Brochures

Related products

Certificates of analysis (COAs)


Safety data sheet (SDS)

For additional assistance, contact applicationsupport@idtdna.com.

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PrimeTime® Gene Expression Master Mix delivers consistent results that are as good or better than the other commercially available master mixes tested. Cq values using the IDT master mix were approximately 2–4 Cqs earlier in this representative experiment.

IDT qPCR master mix is as good or better than others

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).

As good or better than other commercial master mixes

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Obtain successful results with singleplex and duplex amplification using PrimeTime Gene Expression Master Mix and qPCR assays from IDT or other suppliers. All assays exhibited 90–110% PCR efficiency with R2 >0.99.

IDT master mix, PrimeTime qPCR Assays

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 (101–10copies) as template, were run on a 7900HT Real-Time PCR System (Thermo Fisher Scientific).
HPRT Assay ID: Hs.PT.58v.45621572 and GUSB Assay ID: Hs.PT.58v.27737538.

IDT master mix, inventoried qPCR assay (Supplier A)

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).

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

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PrimeTime® Gene Expression Master Mix performs well under standard or fast cycling conditions (Figures 1–2) 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.

PCR efficiency distribution (standard and fast cycling)

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.

Consistent Cq Values (standard and fast cycling conditions)

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.

Standard or fast cycling conditions

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You can expect consistent performance, even when using different batches 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.

Batch-to-batch consistency (0–24 hr at room temperature; HPRT/FAM assay)

A. PrimeTime® HPRT qPCR Assay (FAM-labeled probe; Assay ID: Hs.PT.58v.45621572)

Batch-to-batch consistency (0–24 hr at room temperature; GUSB/HEX assay)

B. PrimeTime® GUSB qPCR Assay (HEX-labeled probe; Assay ID: Hs.PT.58v.27737538)
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.

Batch-to-batch consistency (0–24 hr at room temperature)

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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 heat stress test (4 or 8 hours at 55°C).

Overlay of amplification plot (master mix heated to 55°C for 0, 4, 8 hr; GUSB/HEX assay)

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.

Temperature stability (4 and 8 hr at 55°C)