Demand the most from your qPCR experiments! Flexible, affordable, gold-standard performance.
PrimeTime qPCR Assays
Researchers who use intercalating dyes, like SYBR® Green I, for qPCR experiments typically do so for the advantage they bring in lowered price and reduced turnaround time. Custom primers are inexpensive and are received quickly, and intercalating dyes replace the need for an assay-specific modified probe. However, the disadvantages to using these reagents are significant. First, intercalating dyes bind to any double-stranded DNA, not just to the desired amplification product. This means that the signal generated could be from cross-reacting genes, primer-dimers, or other non-specific amplification. Second, effective primers are difficult to properly design and extra melt curves and gels are required to prove specific target amplification.
5’ nuclease assays, such as PrimeTime® qPCR Assays, include the addition of a hydrolysis probe; this assay dependent probe increases specificity by only generating signal when the correct target is amplified. This increase in specificity allows for simpler assay design and a higher success rate. In addition, because the fluorescence is specific to the gene target, you can use probes with different dyes to measure more than one target in a single tube and save precious sample.
Before PrimeTime qPCR Assays, the disadvantages to using 5’ nuclease assays were cost and synthesis turnaround time of the probe; custom probes take longer to receive and are expensive relative to primers. However, with PrimeTime qPCR Assays, these are no longer a problem. PrimeTime qPCR Assays are estimated to be shipped within 2–4 days of order and are affordably priced. In addition, you have the choice of three different synthesis scales to ensure that you are not paying for unused reactions.
Product focus: qPCR Reagents—everything but your sample
All the reagents you need for successful qPCR assays are now available through IDT.
- Master mix
- Probe-based qPCR assays
- Water and buffer
Free tools for qPCR and PCR assay design
Explore IDT's free, online tools for qPCR probe design and analysis. The design engines for these tools use sophisticated formulas that, for example, take into account nearest neighbor analysis to calculate Tm, and provide the very best qPCR assay designs.
Designing PCR Primers and Probes—Read these general guidelines for designing primers and probes and for choosing target locations for PCR amplification.
Recommended dye combinations for multiplex qPCR—Review these recommendations for selecting dyes for multiplex qPCR that minimize background and avoid overlap of fluorescent signals. Included is a table of compatible dyes for multiplexing on common qPCR instruments and a list of suggested quenchers.
Two Quenchers Are Better Than One!—See the data demonstrating that ZEN™ Double-Quenched Probes in qPCR assays provide increased signal detection and greater assay sensitivity than single-quenched probes, such as probes with BHQ Quenchers. ZEN™ Double-Quenched Probes also make it more feasible to use longer probes due to the resulting lower background fluorescence.
Easily-designed standard curves for qPCR—Learn an easy way to combine control templates/multiple targets onto a single construct and the advantages that can provide for PCR experiments.
Author: Ellen Prediger, PhD, is a scientific writer at IDT.
© 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.