PrimeTime^® qPCR 5′ nuclease probes

Double-quenched probes

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 (Figure 1). 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, Double-Quenched Probes also provide consistently reduced C_q values and improved precision when compared to traditional probes. Use of Double-Quenched Probes can allow you to experience both increased sensitivity and precision in their qPCR experiments.

• ZEN Double-Quenched Probes: ZEN Double-Quenched Probes contain a 5′ fluorophore; 3′ Iowa Black FQ (IBFQ) quencher; and proprietary, internal ZEN quencher. The most commonly supplied 5′ fluorophores includes FAM, HEX™, TET™, Yakima Yellow®, or JOE.
• TAO Double-Quenched Probes: TAO Double-Quenched Probes are available with a 5′ Cy^® 5 fluorophore; 3′ Iowa Black RQ (IBRQ) quencher; and proprietary, internal TAO quencher.

For more information download the PrimeTime Custom qPCR Probes Flyer.

PrimeTime Mini qPCR Probes

The smaller scale and lower price of PrimeTime Mini qPCR probes make them ideal for testing 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.

PrimeTime Eco qPCR Probes

The PrimeTime Eco qPCR Probe is the ideal scale when you 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.

PrimeTime Express qPCR Probes

Express qPCR Probes are HPLC-purified qPCR probes that ship in just one working day! All probes are assessed by mass spectrometry for quality control, with traces provided on our website free of charge. Due to the fast turnaround time, these probes have limited dye-quencher combinations and synthesis scales.

Selecting the correct reporter dye and quencher

• 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 reporter dyes with the least amount of spectral overlap. For assistance in selecting multiplex dye combinations, use our Multiplexing Dye Selection Tool, which helps you select dyes based on what instrument you have. As an additional reference, 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. We have 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 C_q values, improved precision, and enable the use of longer probes for design in AT-rich regions. For more information download the PrimeTime Custom qPCR Probes flyer. We supply 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.
• Summary tables:
  • Instrument compatibility table
  • Dye and quencher wavelength figure

Overview of 5′ nuclease assays

Dye-quencher combinations

To demonstrate the performance of different dye-quencher combinations, we tested a dilution series and found robustness in PCR efficiency and R² values across all dye-quencher combinations available (Figure 1).

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

Figure 1. 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 TaqMan^® Gene Expression Master Mix (Thermo Fisher) under standard cycling conditions. The first four assays were run on the 7900 Fast Real-Time PCR System (Thermo Fisher) and the final assay (Cy5) was run on the LightCycler^® 480 System (Roche).

ZEN Double-Quenched Probes

Achieve maximum quenching for probes as long as 40 bases with ZEN Double-Quenched Probes (Figure 2). Effective quenching means more effective designs, even for AT-rich targets.

TAO Double-Quenched Probes

In qPCR experiments, TAO Double-Quenched Probes exhibit decreased background (Figure 3A) and increased signals (Figure 3B) compared to single-quenched probes.

User guides and protocols

Guides for fluorophore and quencher selection

Brochures and flyers

Frequently asked questions

PrimerQuest^® Design Tool

Design oligos for sequencing, endpoint PCR, and qPCR. Batch design up to 50 sequences at a time. Use our optimized preset design parameters, or customize for your application. Based on the Primer 3 engine.

Multiplex Dye Selection Tool

Select dye combinations compatible with your real-time PCR instrument.

Formulation

We can provide custom formulation for PrimeTime qPCR Probes. Contact Technical and Customer Support for assistance.

GMP and OEM

If you require probes that are approved for use in molecular diagnostic applications, or if you are interested in our OEM services, please click here.