PrimeTime qPCR 5' Nuclease Assays (Probe)

PrimeTime qPCR 5' Nuclease Assays

Primer, Probe mix

Real-Time PCR Probe 2 PCR primers tube Ƽ մϴ.

•• 5' reporter dye FAM, HEX, TET, Cy5, TAMRA
•• 100% QC
•• 5-7 ȿ ϱ single tube
•• PCR ȭ; ideal for many life science research applications
•• ǰ Բ sequence information ִ Specification sheet
•• IDT Real-Time PCR Design Tool
PrimeTime qPCR assays ޵˴ϴ. primers probe premixed Ǿ tube Ƽ lyophilized · ޵Ǹ 500 nM primer 250 nM probe õ 뷮 ̿Ͻ ֽϴ. ֹϽø 1ϸ ޾ƺ ٸ ȸ ޸ ø ؼ 100% QC ̷ ˴ϴ.


No. of Reactions
(20 L)
(FAM-ZEN/Iowa Black FQ)
(other dye-quencher combinations)
Estimated Ship Date Probe (nmoles) Primers (nmoles)2
PrimeTime Mini qPCR Assay
PrimeTime Standard qPCR Assay
PrimeTime XL qPCR Assay

 PrimeTime qPCR 5' Nuclease Assays Reporter Quencher

5' Dye
3' Quencher Mini Standard XL
ZEN/Iowa Black FQ*
ZEN/Iowa Black FQ*
ZEN/Iowa Black FQ*
Iowa Black RQ

Pre-designed PrimeTime qPCR Assay

* 100 % (positive negative control )
* Sequence (ǰ )
* Human, mouse, rat species

Pre-designed PrimeTime qPCR Assay

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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. With 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.
overview of 5 nuclease assays.jpg

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