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PrimeTime™ qPCR Primer Assays
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.
Ordering
- Advanced bioinformatics delivers the design you need with considerations for exon location and number of transcripts identified
- Predesigned sequences will achieve ≥90% efficiency or we will provide an alternative design free of charge
- Test target sequence amplification prior to using more expensive probe-based assays
- Receive primer sequences with all orders
PrimeTime qPCR Primer Assays in tubes
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
PrimeTime qPCR Primer Assays in 96-well plates
Custom assays and predesigned qPCR sequences for human, mouse, and rat targets. Available in standard size, premixed, and shipped dry.
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Product details
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.
PrimeTime qPCR Primer Assays
- The same primer pairs found in the PrimeTime qPCR 5' Nuclease Assays
- Forward and reverse primers, mixed and delivered in a single tube or plate well
- Compatible with SYBR® Green, EvaGreen®, and other intercalating dyes, where no probe is needed
- Available in tubes or 96-well plates
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.
Primer sequences are provided
We provide primer sequences with each order to assist with best practices in research reporting.
- Boosts publication credibility—you can publish your research according to MIQE guidelines
- Helps identify and avoid SNPs
- Facilitates design of multiplex experiments
- Assists with data interpretation and troubleshooting
- Allows you to validate your transcripts
Product data
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%.
Resources
Gene sets for common pathways
Frequently asked questions
How does the PrimeTime™ One-Step RT-qPCR Master Mix compare with one-step master mixes from other manufacturers?
PrimeTime One-Step RT-qPCR Master Mix performance in qPCR experiments is equal to or better than most other master mixes designed for one-step qPCR. Click here to view the representative data.
How long can I leave my qPCR master mix (containing PrimeTime™ Gene Expression Master Mix) at room temperature and still generate consistent results?
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.
Why does the reference dye come in a separate tube from the PrimeTime Gene Expression Master Mix?
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.
Does the PrimeTime™ Gene Expression Master Mix use a hot-start polymerase?
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.
Does IDT offer VIC probes for qPCR?
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.
How should I determine the annealing temperature for a probe-based qPCR assay? Should I select a Tm midway between my primer and probe Tms?
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.
What qPCR products does IDT ship at ambient temperature?
We ship PrimeTime® Gene Expression Master Mix, PrimeTime qPCR assays, and other oligonucleotide products at ambient temperature.
Will phosphothioate (PS) modifications to the ends of PCR primers provide nuclease resistance, and thus a longer half-life for the PCR product when transfected into a cell?
Phosphorothioate bonds (PS bonds) increase oligonucleotide resistance to exonuclease degradation. For single-stranded oligos, nuclease-resistance is highest when every phosphodiester bond is changed to a PS bond. In general we recommend at least 3 PS bonds on both 5' and 3' ends of the oligo, though more PS bonds will give greater exonuclease resistance. Note, however, that PCR primers with PS bonds at both ends will still result in double-stranded (ds) PCR products with PS bonds only at the 5' ends of each product strand. While protected from 5' to 3' nuclease activity, because these dsDNAs lack PS bonds at the 3' strand ends, exonucleases with 3' to 5' activity will still be able to degrade them.
Why do my primers have deleted 3' bases in the amplified fragment when I use a proofreading enzyme for PCR?
Proofreading polymerases (Pfu, Diamond Taq®, etc) have been shown to remove 3' bases from PCR primers (see Nucl Acids Res 20(14):3551-3554, for more information). If you experience this problem, you can protect your primer from degradation by adding one or more phosphorothioate bonds to the 3' end of your primer. Also make sure to add the enzyme last, after the addition of dNTPs, in your PCR to minimize the risk of this issue taking place.
Which dyes are compatible with my thermal cycler?
To determine what dyes are compatible with your instrument, download our Real-time qPCR assay design guide list.
Which dyes are available for use with the IDT ZEN quencher?
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.
Where on a nucleotide are Cy dyes attached?
5′ and 3' Cy dyes are attached to the hydroxyl group of the ribose via a phosphodiester bond.
Internal Cy dyes are attached to the backbone via the phosphodiester bonds of the bases between which the dyes occur.
Internal Cy dyes are attached to the backbone via the phosphodiester bonds of the bases between which the dyes occur.
When will my oligos be shipped?
SameDay® oligos ordered online before 3:00 pm ET (US and Canada) or 1:00 pm CET (Europe) will ship the same day the order is received. Standard oligos are shipped using overnight delivery within 24 hours of receipt of your order.
For US and Canada orders, Rapid HPLC and HOTplate™ oligos ordered online before 3:00 pm ET and Express Dual-Labeled Probe orders placed online before 2:00 pm ET are shipped the next business day. HPLC- or PAGE-purified oligos are routinely shipped within 3 to 5 working days.
For EU orders, there is no cut-off time for Rapid HPLC and HOTplate oligos and Express Dual-Labeled Probe orders, which are shipped the following business day.
For turnaround time of standard plate orders, contact Customer Care at custcare@idtdna.com or phone (US and Canada only) 1-800-328-2661.
For US and Canada orders, Rapid HPLC and HOTplate™ oligos ordered online before 3:00 pm ET and Express Dual-Labeled Probe orders placed online before 2:00 pm ET are shipped the next business day. HPLC- or PAGE-purified oligos are routinely shipped within 3 to 5 working days.
For EU orders, there is no cut-off time for Rapid HPLC and HOTplate oligos and Express Dual-Labeled Probe orders, which are shipped the following business day.
For turnaround time of standard plate orders, contact Customer Care at custcare@idtdna.com or phone (US and Canada only) 1-800-328-2661.
When preparing the qPCR reaction mix, should I thaw or keep reagents (such as the PrimeTime® Gene Expression Master Mix) on ice?
It is good practice, but not necessary to thaw or keep reagents on ice. We see no difference in performance between reaction mixes prepared with the PrimeTime® Gene Expression Master Mix that were used immediately vs those kept at room temperature for up to 24 hours. PrimeTime Gene Expression Master Mix also shows exceptional temperature stability, even after a heat stress test (up to 8 hr at 55°C). Review the data.
What type of purification should I choose for my qPCR primers?
Standard desalting is the recommended purification for qPCR primers.
What tools does IDT provide to design multiplex qPCR assays?
Use the free IDT OligoAnalyzer® software (www.idtdna.com/scitools) to ensure that primer and probe sets lack hairpins, and homo- and heterodimer interactions. From there, you can link directly to the NCBI BLAST tool to further analyze possible sequence interactions. For help with using the BLAST tool, see Tips for Using BLAST to Locate PCR Primers, in DECODED 1.1, April 2011.
What is the structure of the ZEN internal quencher?
The structure of the ZEN quencher is proprietary to IDT.
What is the stability of fluorescently labeled oligos and their fluorescent modifications?
The stability of fluorescently labeled oligos and fluorophore function is similar to the stability of a standard oligo.
For optimal stability, oligos that are to be stored long term should be stored frozen, at -20°C. If the oligos are going to be resuspended, storage in a TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5–8.0; such as IDTE) is better than using water (non-DEPC treated). The impact from the type of storage medium used is marginal at -20°C, but becomes more important at higher temperatures (see the Technical Report, Oligonucleotide Stability Study, for the data).
With fluorophores, it’s important to remember that exposure to natural light or ozone can negatively impact stability. If fluorescently modified oligos will be exposed to sustained light, protective cover from the lighting source (e.g., using brown tubes and/or foil covering) should be considered.
For optimal stability, oligos that are to be stored long term should be stored frozen, at -20°C. If the oligos are going to be resuspended, storage in a TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5–8.0; such as IDTE) is better than using water (non-DEPC treated). The impact from the type of storage medium used is marginal at -20°C, but becomes more important at higher temperatures (see the Technical Report, Oligonucleotide Stability Study, for the data).
With fluorophores, it’s important to remember that exposure to natural light or ozone can negatively impact stability. If fluorescently modified oligos will be exposed to sustained light, protective cover from the lighting source (e.g., using brown tubes and/or foil covering) should be considered.
What is the best way to purify PCR products?
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.
What dyes should I select for my qPCR assay?
IDT offers a free Real-time qPCR assay design guide that contains additional information on available dyes, and instrument compatibility.
*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.
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