Custom qPCR Probes

Generate reliable, high-quality gene expression analysis data for a broad range of sample types with our double- and single-quenched PrimeTime® qPCR Probes.

What you can expect from PrimeTime qPCR Probes:

  • Choose from a wide variety of dyes and quenchers, including several license-free combinations.
  • Use robust ZEN™ or TAO™ Double-Quenched Probes when you need:
    • Reduced crosstalk and amount of sample needed
    • Successful multiplex experiments
    • To target A-T rich regions requiring long probes (up to 40 bases with ZEN probes)
  • Begin your project sooner with 3–6 day product shipping.

All PrimeTime qPCR Probes are verified by mass spectrometry and HPLC-purified, delivering batch-to-batch consistency to minimize your troubleshooting efforts.


Custom qPCR probes

If you know your sequence, choose from a wide variety of synthesis scales, dyes, and quenchers, including several license-free combinations.

Express qPCR Probes

Ready for shipment in one business day. If you know your sequence, select from FAM/ZEN™/Iowa Black® FQ or FAM/Black Hole Quencher®-1.

PrimerQuest® Design Tool

Generate qPCR probes targeting any sequence that is downloaded from Genbank, uploaded as an Excel file, or entered manually.

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Choose from a wide variety of synthesis scales, dyes, and quenchers, including several license-free combinations. Prices and estimated turnaround times depend on the degree of complexity. For more information on dyes and quenchers, visit the Support tab.



PrimeTime® qPCR Probes—Standard Level

The estimated turnaround time for Standard Level Probes is 3–5 business days. The minimum guarantees are listed for probes that are 15–35 bases in length.
5' Reporter Dye(s)QuencherSynthesis Scale / Minimum Guarantee
100 nmol / 15 nmol250 nmol / 25 nmol1 umol / 50 nmol
FAMZEN / Iowa Black FQ *$195.00 USD$275.00 USD$415.00 USD
5' Reporter Dye(s)QuencherSynthesis Scale / Minimum Guarantee
100 nmol / 10 nmol250 nmol / 25 nmol1 umol / 50 nmol
FAMTAMRA$215.00 USD$300.00 USD$460.00 USD
Black Hole Quencher 1$215.00 USD$300.00 USD$460.00 USD
HEXZEN / Iowa Black FQ *$245.00 USD$350.00 USD$520.00 USD
Black Hole Quencher 1$265.00 USD$380.00 USD$575.00 USD
TETZEN / Iowa Black FQ *$245.00 USD$350.00 USD$520.00 USD
Black Hole Quencher 1$265.00 USD$380.00 USD$575.00 USD
YAKZEN / Iowa Black FQ *$245.00 USD$350.00 USD$520.00 USD

* ZEN/Iowa Black FQ is a double-quenched probe which provides superior performance compared to traditional single-quenched probes. For more information download the ZEN Double‑Quenched Probe Overview.



PrimeTime® qPCR Probes—Complex Level

The estimated turnaround time for Complex Level Probes is 4–6 business days. The minimum guarantees are listed for probes that are 15–35 bases in length.
5' Reporter Dye(s)QuencherSynthesis Scale / Minimum Guarantee
100 nmol / 2 nmol250 nmol / 8 nmol1 umol / 20 nmol
FAMTAMRA NHS EsterN/A$450.00 USD$640.00 USD
MAX NHSZEN / Iowa Black FQ *$445.00 USD$515.00 USD$765.00 USD
Black Hole Quencher 1$445.00 USD$515.00 USD$765.00 USD
Cy3Iowa Black RQ$225.00 USD$260.00 USD$475.00 USD
Black Hole Quencher 2$345.00 USD$395.00 USD$600.00 USD
TEX 615Iowa Black RQ$315.00 USD$365.00 USD$665.00 USD
Black Hole Quencher 2$445.00 USD$515.00 USD$765.00 USD
Cy5Iowa Black RQ$225.00 USD$260.00 USD$475.00 USD
Black Hole Quencher 2$345.00 USD$395.00 USD$600.00 USD
TAO / Iowa Black RQ *$225.00 USD$260.00 USD$475.00 USD
TYE 563Iowa Black RQ$315.00 USD$365.00 USD$665.00 USD
Black Hole Quencher 2$445.00 USD$515.00 USD$765.00 USD
TYE 665Iowa Black RQ$315.00 USD$365.00 USD$665.00 USD
Black Hole Quencher 2$445.00 USD$515.00 USD$765.00 USD
JOE NHS EsterZEN / Iowa Black FQ *N/A$350.00 USD$520.00 USD
Black Hole Quencher 1N/A$380.00 USD$575.00 USD
TAMRA NHS EsterIowa Black RQN/A$345.00 USD$515.00 USD
Black Hole Quencher 2N/A$455.00 USD$695.00 USD
ROX NHS EsterIowa Black RQN/A$345.00 USD$515.00 USD
Black Hole Quencher 2N/A$455.00 USD$695.00 USD
Texas Red-X NHS EsterIowa Black RQN/A$345.00 USD$515.00 USD
Black Hole Quencher 2N/A$455.00 USD$695.00 USD

* ZEN/Iowa Black FQ and TAO/Iowa Black RQ are double-quenched probes which provide superior performance compared to traditional single-quenched probes.

Custom qPCR Probes

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While traditional probes have approximately 20–30 bases between the fluorophore and the quencher, the internal ZEN™ or TAO™ 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 lower 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.

  • ZEN Double-Quenched Probes

    ZEN Double-Quenched Probes contain a 5’ fluorophore; 3’ IBFQ quencher; and proprietary, internal ZEN quencher. The most commonly supplied 5′ fluorophores includes FAM, Hex™, TET™, MAX™, or JOE. For more information download the ZEN Double‑Quenched Probe Overview.

  • TAO Double-Quenched Probes

    TAO Double-Quenched Probes are available with a 5’ Cy® 5 fluorophore; 3’ IBRQ quencher; and proprietary, internal TAO quencher.

Double-quenched 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 nmol
FAMZEN / Iowa Black FQ *$65.00 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 nmol
FAMZEN / Iowa Black FQ *$79.00 USD

* ZEN/Iowa Black FQ is a double-quenched probe which provides superior performance compared to traditional single-quenched probes. For more information download the ZEN Double‑Quenched Probe Overview.

Small-scale 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 pm CST/3 pm EST. 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 nmol
FAMZEN / Iowa Black FQ *$249.00 USD
Black Hole Quencher-1$270.00 USD

* ZEN/Iowa Black FQ is a double-quenched probe which provides superior performance compared to traditional single-quenched probes. For more information download the ZEN Double‑Quenched Probe Overview.

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, we recommend using reporters dyes with the least amount of spectral overlap. For a list of selected IDT 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 the 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 internal ZEN quencher. TAMRA is also a quencher option for a FAM reporter dye.

Selecting the Correct Reporter Dye and Quencher

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Dye Calibration Protocols for Common Instruments

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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

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In qPCR experiments, TAO™ Double-Quenched Probes exhibit decreased background (A) and increased signals (B) compared to single-quenched probes.

TAO Double-Quenched Probe
TAO Double-Quenched Probe

Increased signal detection and assay sensitivity from TAO™ Double-Quenched Probes. Three qPCR assays using the same primer and probe sequences were designed to amplify HPRT. The assays differed by the quenchers on the probes—the 5′ Cy® 5 fluorophore was combined with either TAO™ and Iowa Black® RQ (IBRQ) quenchers (blue, double-quenched probe), an IBRQ quencher (pink, single-quenched probe), or a Black Hole Quencher-2 (BHQ2) quencher (green, single-quenched probe). An IDT gBlocks® Gene Fragment encoding HPRT sequences was used as template in a 10 µL PCR on a BioRad CFX384 using Immolase™ DNA Polymerase (Bioline) and the following cycling conditions: 10 min. 95°C; 45 × (15 sec. 95°C, 1 min. 60°C).

TAO™ Double-Quenched Probes