gene_expression

PrimeTime® qPCR 5′ nuclease probes

Generate reliable, high-quality gene expression analysis data with double- and single-quenched fluorescent hydrolysis probes.

PrimeTime qPCR Probes are 5′ nuclease probes, available with an assortment of reporter-dye combinations that are compatible with common qPCR instruments and that include several license-free combinations.

  • Reduce background signals, even with longer probes, when using ZEN™ or TAO™ Double-Quenched Probes
  • Successfully multiplex with reduced crosstalk using double-quenched probes
  • Achieve high efficiency of qPCR under fast or standard cycling conditions
  • Minimize troubleshooting: PrimeTime qPCR Probes are verified by mass spectrometry* and HPLC-purified

* With the exception of mixed base oligos, which could potentially represent multiple sequences and therefore cannot be accurately evaluated by ESI mass spectrometry.

Ordering

Choose from a wide variety of synthesis scales, dyes, and quenchers, including several license-free options. Prices and estimated turnaround times depend on the degree of complexity. For more information on dyes and quenchers, see Product details.

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 *N/A$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 PrimeTime Custom qPCR Probes Flyer.

PrimeTime qPCR Probes—complex level

The estimated turnaround time for complex level probes is 5–7 business days. The minimum guarantees are listed for probes of 15–35 bases.

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 is a double-quenched probe, which provides superior performance compared to traditional single-quenched probes. For more information, download the PrimeTime Custom qPCR Probes Flyer.

PrimeTime Mini qPCR Probes

Available with a 5′ FAM and the option of a 3′ IBFQ quencher alone or in combination with the internal ZEN quencher. EStimated time to shipment 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

Shipped within 3–5 business days.

5' Reporter Dye(s)Quencher(s)Delivery Amount
2.5 nmol
FAMZEN / Iowa Black FQ *$79.00 USD

PrimeTime Express qPCR Probes

HPLC purified. Availabile for lengths of 18–35 bases. Shipped dry. Minimum guarantee of 5 nmol.

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 PrimeTime Custom qPCR Probes Flyer.

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 Cq 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.

Figure 1. Double-quenched probe showing position of internal ZEN Quencher.

  • 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 Cq 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:

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.

primetime-5'-nuclease-overview

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 Amplification Curve - FAM / Iowa Black FQ Amplification Curve - FAM / TAMRA Amplification Curve - HEX / Iowa Black FQ Amplification Curve - TET / Iowa Black FQ Amplification Curve - Cy5 /Iowa Black RQ
Standard curve Standard Curve - FAM / Iowa Black FQ Standard Curve - FAM / TAMRA Standard Curve - HEX / Iowa Black FQ Standard Curve - TET / Iowa Black FQ Standard Curve - Cy5 /Iowa Black RQ
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.

Figure 2. Only ZEN Double-Quenched Probes maintain low background signal with increasing probe length. Probes of 2 lengths (20 or 40 bases) with 5 different quenchers were compared in 10 singleplex qPCRs. Six replicate reactions with each probe type were run with 50 ng of cDNA and the TaqMan Gene Expression Master Mix (Thermo Fisher) under standard cycling conditions on the Applied Biosystems 7900HT system. Key: IBFQ = Iowa Black FQ Quencher; BHQ®-1 = Black Hole Quencher®-1 (Biosearch Technologies); Eclipse® = Eclipse quencher (ELITech Group).

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.

Figure 3. 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). A gBlocks® Gene Fragment (IDT) encoding HPRT sequences was used as template in a 10 µL PCR on a CFX384 instrument (BioRad) using Immolase™ DNA Polymerase (Bioline) and the following cycling conditions: 10 min. 95°C; 45 × (15 sec. 95°C, 1 min. 60°C).

User guides and protocols

PDF tag Dye calibration—AB 7300 and 7500/7500 Fast (139 KB)
PDF tag Dye calibration—AB 7300 and 7500/7500 Fast (139 KB)
PDF tag PrimeTime qPCR Assay Plate resuspension (507 KB)
PDF tag PrimeTime qPCR Assay Plate resuspension (507 KB)
PDF tag Dye calibration—AB 7900 Fast (179 KB)
PDF tag Dye calibration—AB 7900 Fast (179 KB)

Guides for fluorophore and quencher selection

Instrument compatibility table
Dye and quencher wavelength chart

Brochures and flyers

PDF tag PrimeTime qPCR Assays (536 KB)
PDF tag PrimeTime qPCR Assays (536 KB)
PDF tag PrimeTime Custom qPCR Probes (672 KB)
PDF tag PrimeTime Custom qPCR Probes (672 KB)
PDF tag PrimeTime Gene Expression Master Mix (668 KB)
PDF tag PrimeTime Gene Expression Master Mix (668 KB)

Frequently asked questions

All related FAQs

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.