Premixed primer pairs for analyzing gene expression using intercalating dyes.
PrimeTime qPCR Primer Assays provide a primer pair designed for real-time PCR using intercalating dyes, such as SYBR® Green (Thermo Fisher Scientific) or EvaGreen® (Biotium) dyes. Predesigned sequences for human, mouse, or rat are designed with advanced bioinformatic and thermodynamic sequence analytics and for easy selection. Custom assays may be created for any sequence from any species using the PrimerQuest™ Tool.
Available in standard size, premixed, and shipped dry.
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1 Available for human, mouse, and rat targets (for assay configuration, choose intercalating dyes, primers only)
2 Choose design: qPCR–2 primers–intercalating dyes
Custom assays and predesigned qPCR sequences for human, mouse, and rat targets. Available in standard size, premixed, and shipped dry.
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PrimeTime qPCR Primer Assays can be used with SYBR® Green (Thermo Fisher Scientific), EvaGreen® (Biotium), and other intercalating dyes, where no probe is needed. PrimeTime qPCR Primer Assays are available in standard size, premixed, normalized to 5 nmol per primer, and shipped dried down. Primer sequences are provided upon order.
Predesigned sequences are designed using a proprietary algorithm. The bioinformatic calculations optimizes oligo melting temperature (Tm), base composition, oligo length, etc., and accounts for factors such as SNPs (based on current NCBI RefSeq releases), off-target amplification, splice variants, and secondary structures.
Guarantee: Predesigned sequences will achieve 90% efficiency or better, or we will provide an alternative design free of charge. For more information, please contact us.
Custom primer assays can also be designed for other species using the PrimerQuest™ Tool. This tool may be used to design oligos for endpoint PCR, qPCR, and Sanger sequencing. Use our preset design parameters for PCR and qPCR or customize parameters for your application. The PrimerQuest Tool is based on the Primer3 engine.
For commonly studied pathways in human, mouse, and rat species, we have suggested gene sets (see Resources section below) that can be used with our PrimeTime plate ordering system.
For help with assay design, contact us.
We provide primer sequences with each order to assist with best practices in research reporting.
Figure 1. PrimeTime qPCR Primer Assays yield the same high efficiency whether used with intercalating dyes or probes. Amplification of 5 sequential 4-fold dilutions of cDNA using PrimeTime qPCR Primer Assays (with SYBR® Green dye [Thermo Fisher Scientific]) or the PrimeTime qPCR 5′ Nuclease Assay (with dual-labeled probe) targeting human 3-oxoacid CoA transferase 1 (OXCT1) (NM_000436).
Figure 2. PrimeTime qPCR Primer Assays have an average reaction efficiency >90%. Sixty randomly selected PrimeTime qPCR Primer Assays and 15 PrimeTime qPCR Primer Assays for endogenous control genes used with Brilliant III Ultra-Fast SYBR® Green qPCR Master Mix (Agilent) were analyzed over 5 sequential 4-fold dilutions (50–0.195 ng/reaction) of cDNA prepared from Universal Human Reference RNA (Agilent). Reactions were run on the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific) using PCR cycling conditions: 3 min 95°C; 45 x (5 sec 95°C, 15 sec 60°C). Average reaction efficiencies for the assays tested exceeded 98%.
The stability of PrimeTime Gene Expression Master Mix makes it ideal for overnight PCR runs and high throughput experiments using robotic liquid handling systems.
Reliable results have been obtained when the qPCR was run directly after setup or after the reactions were maintained at room temperature for up to 72 hr.
Download the white paper, Ambient shipping of PrimeTime Gene Expression Master Mix for more data.
We provide a separate tube of reference dye so that you may use the PrimeTime™ Gene Expression Master Mix on reference dye-dependent or dye-independent instruments.
Also, the separate tube of reference dye provides a way to add a high or low dye concentration to the master mix, depending on the requirements of your instrument.
Yes, it uses an antibody-mediated, hot-start polymerase. During the initial denaturation step of qPCR cycling, polymerase activity is unblocked when the polymerase is released from the antibody.
This hot-start polymerase enhances reaction precision of the qPCR by avoiding non-specific amplification of DNA and primer dimer formation. It is therefore critical to follow the cycling conditions provided in the PrimeTime Gene Expression Master Mix Protocol.
Yes, IDT offers the SUN™ dye, which is our product offering for the VIC® dye (ThermoFisher).
More information about probes labeled with the SUN fluorophore can be found on the PrimeTime™ qPCR probes page.
When performing qPCR it is ideal to have your probe Tm about 5-10 degrees higher than your primer Tms. The annealing temperature should be set 3-5 degrees lower than the lowest primer Tm.
Use this as a general guideline, but note that optimization may still be necessary.
Currently, the IDT ZEN™ quencher is available with FAM, HEX, TET, Yakima Yellow® (ELITech Group), JOE™ (ThermoFisher), MAX™, and SUN™ fluorophores. Double-quenched probes have significantly lower background fluorescence, especially for longer probes.
The ZEN quencher is available on PrimeTime™ qPCR Probes and on the probes supplied with PrimeTime qPCR Probe Assays. For any special oligonucleotide requests that include the ZEN quencher but are not found on our website (non-catalog requests), please contact applicationsupport@idtdna.com.
For more information, see Double-quenched probes increase qPCR sensitivity and precision.
ZEN, SUN, MAX, and PrimeTime are trademarks of IDT.
For most applications, it is best to purify PCR products by gel electrophoresis.
This simple method not only purifies the final PCR product but can be a valuable troubleshooting tool as well since nonspecific PCR products, primer-dimer products, negative amplification, and other elements can easily be identified by gel analysis.
*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.