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PrimeTime® qPCR Probes

PrimeTime qPCR DNA probes are non-extendable oligonucleotides labeled with a 5’ fluorescent reporter and a 3’ quencher dye licensed for use in 5’ nuclease qPCR assays (with companion dye and quencher licenses).

PrimeTime® qPCR Probes

  • Traditional dual-labeled probes are available with a wide variety of dyes and quenchers.
  • Choose IDT proprietary ZEN™ Double-Quenched Probes for superior performance compared to traditional dual-labeled probes.
  • Ultra-small-scale Mini Probes delivered at 0.5 nmole allow researchers to conduct initial screening at low cost.
  • Eco Probes are delivered at 2.5 nmoles normalized yield, sufficient for ~500 reactions for gene expression analysis.
  • Express Probes are ready for shipment in one business day.

ZEN™ Internal Quencher

  • The ZEN™ quencher enables the production of Double-Quenched Probes that provide less background and increased signal!
  • Double-Quenched Probes are available with 5’ FAM, TET, HEX, MAX, or JOE fluorophores, a 3’ IBFQ quencher, and an internal ZEN™ quencher.
  • ZEN™ Double-Quenched Probes display consistently earlier Cq values and improved precision when compared to traditional probes. 

For more information download the ZEN Double-Quenched Probe Overview.

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PrimeTime® qPCR probes are available with a variety of reporter/quencher combinations and synthesis scales. More information on dyes and quenchers can be found under the Support tab. 

The prices and estimated turnaround time for these probes will depend on the degree of complexity. Probes at the Standard Level will ship in approximately 3–5 business days while probes at the Complex Level will ship in approximately 4–6 business days. The minimum guarantees are listed for probes that are 15–35 bases in length.

If you require qPCR probes for an application other than the 5’ Nuclease Real-Time PCR assay, please use the DLP builder.

PrimeTime® qPCR Probes - Standard Level

5' Reporter Dye(s)QuencherSynthesis Scale / Minimum Guarantee
100 nmole / 15 nmoles250 nmole / 25 nmoles1 umole / 50 nmoles
FAMZEN / Iowa Black FQ *$195.00 USD$275.00 USD$415.00 USD
5' Reporter Dye(s)QuencherSynthesis Scale / Minimum Guarantee
100 nmole / 10 nmoles250 nmole / 25 nmoles1 umole / 50 nmoles
FAMTAMRA$195.00 USD$275.00 USD$415.00 USD
Black Hole Quencher 1$195.00 USD$275.00 USD$415.00 USD
HEX
TET
ZEN / Iowa Black FQ *$245.00 USD$350.00 USD$520.00 USD
Black Hole Quencher 1$245.00 USD$350.00 USD$520.00 USD

*ZEN/Iowa Black FQ is a Double-Quenched Probe which provides superior performance to traditional dual-labeled probes. For more information download the ZEN Double‑Quenched Probe Overview.

The estimated turnaround time for Standard Level Probes is 3–5 business days.

PrimeTime® qPCR Probes - Complex Level

5' Reporter Dye(s)QuencherSynthesis Scale / Minimum Guarantee
100 nmole / 2 nmoles250 nmole / 8 nmoles1 umole / 20 nmoles
FAMTAMRA NHS EsterN/A$415.00 USD$580.00 USD
MAX NHSZEN / Iowa Black FQ *$315.00 USD$365.00 USD$545.00 USD
Black Hole Quencher 1$315.00 USD$365.00 USD$545.00 USD
Cy3
TEX 615
Cy5
TYE 563
TYE 665
Iowa Black RQ$225.00 USD$260.00 USD$475.00 USD
Black Hole Quencher 2$315.00 USD$365.00 USD$545.00 USD
JOE NHS EsterZEN / Iowa Black FQ *N/A$420.00 USD$630.00 USD
Black Hole Quencher 1N/A$420.00 USD$630.00 USD
TAMRA NHS Ester
ROX NHS Ester
Texas Red-X NHS Ester
Iowa Black RQN/A$345.00 USD$515.00 USD
Black Hole Quencher 2N/A$420.00 USD$630.00 USD

The estimated turnaround time for Complex Level Probes is 4–6 business days.

PrimeTime® qPCR Probes

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PrimeTime® Mini qPCR Probes

The PrimeTime® Mini qPCR probes are probes available on a small scale. The smaller scale and lower price makes them ideal for trying out a new probe, for screening the expression levels of many genes, or for testing the conversion to FAM/IBFQ and FAM/ZEN/IBFQ from other FAM-related quenchers. Mini probes are offered with a 5’ FAM and the option of a 3’ IBFQ quencher alone or in combination with the internal ZEN™ quencher. The estimated turnaround time is 3–5 days.

5' Reporter Dye(s)Quencher(s)Delivery Amount
0.5 nmoles
FAMZEN / Iowa Black FQ *$60.00 USD

PrimeTime® Eco qPCR Probes

The PrimeTime® Eco qPCR Probe is the ideal scale for researchers who need to perform ~500 reactions for gene expression analysis. The combination of medium scale and low cost is ideal for initial screening of large sample sets. Eco qPCR Probes are available as double-quenched probes, with 5' FAM and a 3' IBFQ quencher in combination with an internal ZEN™ quencher. Eco probes are shipped within 3–5 business days.
5' Reporter Dye(s)Quencher(s)Delivery Amount
2.5 nmoles
FAMZEN / Iowa Black FQ *$79.00 USD

* ZEN/Iowa Black FQ is a Double-Quenched Probe which provides superior performance to traditional dual-labeled probes. For more information download the ZEN Double‑Quenched Probe Overview.

The turnaround time from order to ship for PrimeTime Eco qPCR Probes is 3–5 business days.

PrimeTime® Mini and Eco qPCR Probes

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Express qPCR probes allow researchers the opportunity to have their qPCR probes ship in just one working day! All probes are HPLC-purified and assessed by mass spectrometry for quality control, with the trace provided free on our website. Due to the fast turnaround time, these probes are limited to select dye-quencher combinations and synthesis scales. All Express probes must be 18–35 bases. Probes are shipped dry and have a minimum guarantee of 5 nmoles. 

All orders for Express qPCR Probes must be placed online by 2:00 pm ET. Probes are shipped the next business day for delivery by 10:30 am the following business day. Additional shipping and handling fees for expedited service apply.


PrimeTime® Express qPCR Probes

5' Reporter Dye(s)Quencher(s)Synthesis Scale / Minimum Guarantee
100nm / 5 nmoles
FAMZEN / Iowa Black FQ *$249.00 USD
Iowa Black$229.00 USD
Black Hole Quencher-1$249.00 USD

*ZEN/Iowa Black FQ is a Double-Quenched Probe which provides superior performance to traditional dual-labeled probes. For more information download the ZEN Double‑Quenched Probe Overview.

The turnaround time from order to ship for Express Dual Labeled Probes is 1 business day.

Express qPCR Probes

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In step one, 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.

In step two, the polymerase extends from the primers and begins DNA synthesis.

In step three, 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. In step four, this fluorescence is detected by the real time instrument. These steps are repeated for each PCR cycle and allow detection of specific products. With intercalation 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.

Overview of 5′ Nuclease Assays

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Reporter Dyes

The correct reporter dye will depend on the type of instrument you are using and the compatibility of the dye with the instrument. Please see the Instrument Compatibility Table for a list of reporter dyes compatible with common instrumentation. 

For multiplexing applications, it is recommended that reporters dyes with the least amount of spectral overlap be selected. For a complete list of IDT’s dyes and quenchers please see the Dye and Quencher Wavelength Figure and the Instrument Compatibility Table 

Quenchers

Traditional dark quenchers absorb broadly and do not emit light which allows for the use of multiple reporter dyes with a given quencher. This characteristic allows for expanded options for multiplex assays. Dark quenchers simplify detection which makes them compatible with a broad range of image analysis instruments. 

IDT has developed an internal ZEN quencher that enables to production of Double-Quenched Probes which have less background and more signal. The Double-Quenched Probes contain a 5’ FAM fluorophore, a 3’ IBFQ quencher, and an internal ZEN quencher. These Double-Quenched Probes are an improvement over traditional dual-labeled probes and have consistently low background, reduced Cq values, improved precision, and enable the use of longer probes for design in AT-rich regions. For more information download the ZEN Double-Quenched Probe Overview

IDT supplies commonly used dark quenchers as well as the proprietary dark quenchers, Iowa Black FQ, Iowa Black RQ, and the newly developed internal ZEN quencher. TAMRA is also a quencher option for a FAM reporter dye. Related Information

Selecting the Correct Reporter Dye and Quencher

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Dye Calibration Protocols for Common Instruments

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IDT has developed the internal ZEN™ quencher which enables the production of Double-Quenched Probes with less background and increased signal! Double-Quenched Probes contain a 5’ FAM fluorophore, a 3’ IBFQ quencher, and an internal ZEN quencher. While traditional probes have approximately 20–30 bases between the fluorophore and the quencher, the internal ZEN quencher decreases that length to only 9 bases. This shortened distance, particularly when combined with the traditional 3’ end quencher, leads to a much more thorough quenching with much less background and enables the use of much longer probes for designing in AT-rich target regions. In addition to the significantly decreased background, the Double-Quenched Probes also have consistently reduced Cq values and improved precision when compared to traditional probes. Use of the Double-Quenched Probes can allow users to experience both increased sensitivity and precision in their qPCR experiments.

For more information download the ZEN Double-Quenched Probe Overview.

ZEN™ Double-Quenched Probes

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IDT and Biogazelle are working together to provide users of PrimeTime® qPCR products a solution for qPCR data analysis. Receive 35% off the list price of a premium qbasePLUS license from Biogazelle.

Biogazelle is the real-time PCR data analysis company, built upon extensive experience in real-time PCR experiment design, assay development, and data-analysis. Groundbreaking publications on normalization of gene expression and data analysis by Biogazelle founders, Jo Vandesompele and Jan Hellemans, have been cited more than 3000 times.

qbasePLUS software from Biogazelle is a qPCR analysis package that allows direct import of data from qPCR instruments from a variety of manufacturers. The software is based on proven geNorm and qBase technology and enhanced with proprietary algorithms and time-saving features. These algorithms remove data errors, normalize data to one or more reference genes, and correct inter-run variation using inter-run calibrators. qbasePLUS software offers statistical tools for qPCR data analysis and graphical presentation tools for viewing analyzed data. It is the most powerful, flexible, and user-friendly real-time PCR data analysis software available. qbasePLUS is compatible with Windows, Mac, and Linux operating systems and meets MIQE guidelines.

Biogazelle offers a variety of other services to help investigators design and implement qPCR experiments, including educational materials and courses. For a complete overview of Biogazelle wet lab and data mining services, visit www.biogazelle.com.

Data Analysis Recommendations

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To demonstrate the performance of different Dye-Quencher combinations, IDT tested a dilution series and found robustness in PCR efficiency and R2 values across all Dye-Quencher combinations available. 

Dye-Quencher Combination FAM / Iowa Black FQ FAM / TAMRA HEX / Iowa Black FQ TET / Iowa Black FQ Cy5 /Iowa Black RQ
Amplification Curve
Standard Curve
Efficiency 95.7% 95.1% 94.7% 94.5% 98.0%
Correlation Coefficient (R²) >0.999 >0.999 >0.999 >0.999 >0.998

Demonstrated Assay Performance with Multiple Dye Quencher Combinations. A plasmid dilution series of the CSK (c-src tyrosine kinase) Assay was used to test different dye quencher combinations. The data illustrates robustness in PCR efficiency and R2 values close to one across all dye/quenchers available for PrimeTime® Assays. All reactions were run with Gene Expression Master Mix (Applied Biosystems) under standard cycling conditions. The first four assays were run on the 7900 Fast Real-Time PCR System (Applied Biosystems) and the final assay (Cy5) was run on the LightCycler® 480 System (Roche).

 

Dye-Quencher Combinations