gene_expression

PrimeTime qPCR Probes

Generate reliable, high-quality gene expression analysis data with double- or 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.

  • Use MGB (minor groove binding) probes for stabilized probe-target hybridization and increased melting temperature (Tm), allowing for shorter probes with better allelic discrimination
  • Successfully multiplex with reduced crosstalk using double-quenched probes
  • Achieve high efficiency qPCR under fast or standard cycling conditions
  • Minimize troubleshooting: PrimeTime qPCR Probes are verified by mass spectrometry* and are HPLC-purified
  • Select Mini or Eco qPCR probes for digital PCR (dPCR) platforms
  • Choose from a variety of shipping options to meet your timeline or budget

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

Ordering

Determine your preferred shipping option—Express, Small scale (Eco/Mini), Quick ship, Standard ship. Choose from a wide variety of synthesis scales, dyes, and quenchers. Prices and estimated turnaround times depend on the degree of complexity or yield. 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) / Emission (nm)QuencherMinimum Yield GuaranteeMinimum Yield GuaranteeMinimum Yield Guarantee
15 nmol25 nmol50 nmol
FAM
520
ZEN / Iowa Black FQ *$195.00 USD$275.00 USD$415.00 USD
5' Reporter Dye(s) / Emission (nm)QuencherSynthesis Scale / Minimum Guarantee
100 nmol / 10 nmol250 nmol / 25 nmol1 umol / 50 nmol
FAM
520
TAMRA$215.00 USD$300.00 USD$460.00 USD
Black Hole Quencher 1$215.00 USD$300.00 USD$460.00 USD
TET
539
ZEN / 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
YAK
549
ZEN / Iowa Black FQ *N/A$350.00 USD$520.00 USD
HEX
555
ZEN / 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

Black Hole Quencher is a trademark of Biosearch Technologies, Inc.

* 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) / Emission (nm)QuencherMinimum Yield GuaranteeMinimum Yield GuaranteeMinimum Yield Guarantee
2 nmol8 nmol20 nmol
FAM
520
TAMRA NHS EsterN/A$450.00 USD$640.00 USD
MGB-NFQ
SUN
554
ZEN / Iowa Black FQ *$195.00 USD$275.00 USD$415.00 USD
Iowa Black RQ
MGB-NFQ
HEX
555
MGB-NFQ
JOE NHS Ester
555
ZEN / Iowa Black FQ *N/A$350.00 USD$520.00 USD
Black Hole Quencher 1N/A$380.00 USD$575.00 USD
MAX NHS
556
ZEN / 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
TYE 563
563
Iowa Black RQ$315.00 USD$365.00 USD$665.00 USD
Black Hole Quencher 2$445.00 USD$515.00 USD$765.00 USD
Cy3
564
Iowa Black RQ$225.00 USD$260.00 USD$475.00 USD
Black Hole Quencher 2$345.00 USD$395.00 USD$600.00 USD
TAMRA NHS Ester
583
Iowa Black RQN/A$345.00 USD$515.00 USD
Black Hole Quencher 2N/A$455.00 USD$695.00 USD
TEX 615
613
Iowa Black RQ$315.00 USD$365.00 USD$665.00 USD
Black Hole Quencher 2$445.00 USD$515.00 USD$765.00 USD
Texas Red-X NHS Ester
617
Iowa Black RQN/A$345.00 USD$515.00 USD
Black Hole Quencher 2N/A$455.00 USD$695.00 USD
TYE 665
665
Iowa Black RQ$315.00 USD$365.00 USD$665.00 USD
Black Hole Quencher 2$445.00 USD$515.00 USD$765.00 USD
Cy5
668
Iowa 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

Black Hole Quencher is a trademark of Biosearch Technologies, Inc.

* 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 turnaround time for probes is 3–6 days.

5' Reporter Dye(s) / Emission (nm)Quencher(s)Delivery AmountDelivery Amount
0.5 nmol2.5 nmol
FAM
520
ZEN / Iowa Black FQ *$65.00 USD$79.00 USD
MGB-NFQ
SUN
554
ZEN / Iowa Black FQ *
MGB-NFQ
HEX
555
ZEN / Iowa Black FQ *
MGB-NFQ
Cy5
668
TAO/ Iowa Black RQ *

PrimeTime Eco qPCR Probes

Estimated turnaround time for probes is 3–6 days.

5' Reporter Dye(s) / Emission (nm)Quencher(s)Delivery AmountDelivery Amount
0.5 nmol2.5 nmol
FAM
520
ZEN / Iowa Black FQ *$65.00 USD$79.00 USD
MGB-NFQ
SUN
554
ZEN / Iowa Black FQ *
MGB-NFQ
HEX
555
ZEN / Iowa Black FQ *
MGB-NFQ
Cy5
668
TAO/ Iowa Black RQ *

PrimeTime Express qPCR Probes

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

5' Reporter Dye(s)Quencher(s)Minimum Guarantee
5 nmol
FAM 520ZEN / Iowa Black FQ *$95.00 USD
Black Hole Quencher-1$150.00 USD

Black Hole Quencher is a trademark of Biosearch Technologies, Inc.

* 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

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. These probes are ideal for digital PCR (dPCR) platforms.

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. These probes are ideal for dPCR platforms.

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.

In the United States, PrimeTime MGB Probes, PrimeTime MGB Probes Assays, and MGB SNP Genotyping assays are not limited to human in vitro diagnostic use only. Although a number of other available applications exist, including in vitro diagnostics, commercial, agricultural biotechnology (agbio), and research, each customer who purchases this product is required to obtain their own regulatory approval for any use outside of research.

In other jusidictions, MGB Eclipse Probes are licensed by ELITech for use solely in human in vitro disgnostic end-use applications. Customers need to follow the licensing requirements for their specific juristiction. IDT manufactures MGB Eclipse probes and companion primers under GMP conditions and using ISO certified processes.

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

Guides for fluorophore and quencher selection

Instrument compatibility table
Dye and quencher wavelength chart

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.