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Some applications of this product  may require a license which is not provided by the purchase of this product.

For research use only. 

Cost-effective dye-based qPCR Mix with passive reference dye ROX

  • high sensitivity and specificity
  • excellent efficiency
  • reaction set-up and shipment without dry ice
  • cost-effective solution for wide range of applications

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Catalog number

08-24-00001

Quantity

€ 46.50

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Details

Overview

Description

Dye-based real-time quantitative PCR (qPCR) uses DNA binding dye to evaluate DNA amplification process during PCR. In this mix EvaGreen® double-stranded DNA binding dye that is used instead of more widely used SYBR Green I that has similar fluorescence spectra. Compared to SYBR Green I dye EvaGreen® dye shows higher fluorescence level, high sensitivity for detecting low template concentrations and high stability at room temperature

HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX) is an optimized ready-to-use solution for dye-based real-time quantitative PCR assays on cyclers that require passive reference dye (including high ROX or low ROX reference signal requiering platforms).

Applications

  • Detection and quantification of DNA and cDNA targets
  • Profiling gene expression
  • Microbial detection
  • Viral load determination

Properties

Concentration: 5x

Hot-start: yes, initial activation in 12-15 min

Detection type: dye-based, includes EvaGreen® intercalating dye

Reference dye: based on ROX

Compatible real-time instruments: Cyclers that require ROX reference dye. Check Your cycler!

Mix Components

HOT FIREPol® DNA polymerase: chemically modified FIREPol® DNA Polymerase enabeling hot-start

5x EvaGreen® qPCR buffer with 12. 5 mM MgCl2: 1x PCR solution – 2.5 mM MgCl2

dNTPs: dATP, dCTP, dGTP and dTTP

EvaGreen® dye

Internal reference based on ROX dye

EvaGreen Dye

EvaGreen® is a DNA-binding dye with many features that make it a superior alternative to SYBR® Green I for qPCR. Apart from having similar spectra, EvaGreen® has three important features that set it apart from SYBR® Green I: EvaGreen® has much less PCR inhibition, is an extremely stable dye and has been shown to be non-mutagenic and non-cytotoxic. EvaGreen® is compatible with all common real-time PCR cyclers – simply select the standard settings for SYBR® Green or FAM!

Related Resources

FAQ

What is the difference between dye-based and probe-based systems?

Dye-based detection (e.g., EvaGreen®, SolisGreen®, SYBR® Green) is a cost-effective qPCR option, as it requires only addition of PCR primers. However, the intercalating dye will detect any dsDNA (non-specific amplicons, primer dimers) produced in the reaction. Melt curve analysis performed after the qPCR can be used to verify the specificity of amplification and to check for the presence of non-specific amplification products.

The probe-based qPCR system demonstrates higher specificity compared to dye-based qPCR, because probes only detect the gene of interest. Keep in mind that in probe-based assays, primer dimers and non-specific products will not be detected, however, they may compromise the PCR efficiency. Using probe-based qPCR system, it is possible to distinguish between sequences with high similarity (e.g. single-nucleotide variations). Additionally, probe-based qPCR assays allow for multiplex reactions in one tube, while only a single target can be amplified and measured in a dye-based qPCR.

What is the difference between dye-based and probe-based systems?

Dye-based detection (e.g., EvaGreen®, SolisGreen®, SYBR® Green) is a cost-effective qPCR option, as it requires only addition of PCR primers. However, the intercalating dye will detect any dsDNA (non-specific amplicons, primer dimers) produced in the reaction. Melt curve analysis performed after the qPCR can be used to verify the specificity of amplification and to check for the presence of non-specific amplification products.

The probe-based qPCR system demonstrates higher specificity compared to dye-based qPCR, because probes only detect the gene of interest. Keep in mind that in probe-based assays, primer dimers and non-specific products will not be detected, however, they may compromise the PCR efficiency. Using probe-based qPCR system, it is possible to distinguish between sequences with high similarity (e.g. single-nucleotide variations). Additionally, probe-based qPCR assays allow for multiplex reactions in one tube, while only a single target can be amplified and measured in a dye-based qPCR.

What is the difference between EvaGreen® and SYBR® Green?

Both dyes are DNA intercalating agents that are used to stain dsDNA.

EvaGreen® dye is spectrally similar to FAM or SYBR® Green I, which means that no change in optical settings is required for using an EvaGreen®-based qPCR mix.

EvaGreen® was reported to have several benefits over SYBR® Green I:

  • EvaGreen® dye has less background than SYBR® Green I due to its novel “release-on-demand” DNA-binding mechanism,
  • EvaGreen® is less inhibitory in the PCR reaction than SYBR® Green I, enabling the use of higher dye concentration for maximal signal and better high-resolution DNA melt analysis,
  • EvaGreen® is non-mutagenic, non-cytotoxic and environmentally safe,
  • EvaGreen® is very stable under PCR condition and during routine storage and handling, whereas SYBR® Green I is known to degrade following multiple freeze-thaw cycles and under PCR conditions.
What is the difference between EvaGreen® and SolisGreen® dye?

Both dyes are DNA intercalating agents that are used to stain dsDNA. SolisGreen™ dye is spectrally similar to EvaGreen® allowing detection in the FAM or SYBR® Green I channel (SolisGreen™ excitation maximum 489 nm, emission maximum 515 nm). It has been shown that qPCR mixes based on SolisGreen™ dye have increased performance with low target concentration demonstrating improved precision and less variance between technical replicates.

After first-strand cDNA synthesis, how much cDNA template should I use for my PCR?

Recommended final amount of cDNA sample in downstream PCR reaction is up to one tenth of the final reaction volume. Overload of cDNA sample may compromise the downstream PCR, because cDNA sample may contain reaction components that may inhibit your PCR reaction.

Why do certain kits contain a ROX?

ROX (carboxy-X-rhodamine) is one of the passive reference dyes providing a constant fluorescent signal that is used for signal normalization during the amplification cycles. The emission recorded from a reference dye during the baseline cycles is used to normalize the emission recorded from the reporter (e.g. EvaGreen®, SolisGreen®, SYBR® Green, etc.) in later cycles in ROX-dependent real-time PCR systems (e.g. Applied Biosystems 5700, 7000, 7300, 7700, 7900HT, StepOne™, StepOnePlus™). Reference dye compensates for small fluorescent fluctuations and well-to-well variations that may occur.

In Solis BioDyne qPCR mixes we use technology, based on ROX, that allows us to use same mix for high- and low- ROX requiering cyclers. 

Please check cycler-mix compatibility here if not sure.

What are the storage conditions and stability of Solis BioDyne reagents?

Solis BioDyne products should be stored at -20°C.

Shipping and temporary storage for up to 1 month at room temperature (*1525°C) has no detrimental effects on the quality of Solis BioDyne reagents.

Freeze-thaw stability is tested for each product. Most PCR and qPCR products have passed 30 freeze-thaw cycles with no changes in performance. Specific information is found in Storage and Shipping conditions of each product.

When stored and handled under the recommended conditions, full activity of the reagents is retained until the Expiry Date printed on the tube label.

*World Health Organization (2003). Guidelines for the Storage of Essential Medicines and Other Health Commodities.

Publications

Perinatal exposure to bisphenol A (BPA) impairs neuroendocrine mechanisms regulating food intake and kisspetin system in adult male rats. Evidences of metabolic disruptor hypothesis

C. Stoker et al. / Mol. Cell. Endocrinol. / 2020

Time dependent expression of the blood biomarkers EIF2D and TOX in patients with schizophrenia

J. Gilabert-Juan et al. / Brain. Behav. Immun. / 2019

Dysregulated expression of STAT1, miR‐150, and miR‐223 in peripheral blood mononuclear cells of coronary artery disease patients with significant or insignificant stenosis

Z. Saadatian et al. / J. Cell. Biochem. / 2019
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The impact of sensory and motor enrichment on the epigenetic control of steroidogenic-related genes in rat hippocampus

M. F. Rossetti et al. / Mol. Cell. Endocrinol. / 2019

Multiple adenosine receptor subtypes stimulate wound healing in human EA.hy926 endothelial cells

Z. Bonyanian et al. / Purinergic Signal. / 2019

Yeast applied readthrough inducing system (YARIS): an in vivo assay for the comprehensive study of translational readthrough

P. Beznosková et al. / Nucleic Acids Res. / 2019

Highly Methacrylated Gelatin Bioink for Bone Tissue Engineering

G. Irmak, T. T. Demirtaş, and M. Gumusderelioglu / ACS Biomater. Sci. Eng. / 2019

Effect of neonatal exposure to endosulfan on myometrial adaptation during early pregnancy and labor in rats

R. Alarcón et al. / Mol. Cell. Endocrinol. / 2019

Metabolomic and Transcriptional Analyses Reveal Atmospheric Oxygen During Human Induced Pluripotent Stem Cell Generation Impairs Metabolic Reprogramming

J. Spyrou, D. K. Gardner, and A. J. Harvey / Stem Cells / 2019

Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells

M. Bajzikova et al. / Cell Metab. / 2019

Sex-determining region Y (SRY) attributes to gender differences in RANKL expression and incidence of osteoporosis

K. Kodrič et al. / Exp. Mol. Med. / 2019

Plant circadian rhythms regulate the effectiveness of a glyphosate-based herbicide

F. E. Belbin et al. / Nat. Commun. / 2019

Blockade of miR-140-3p prevents functional deterioration in afterload-enhanced engineered heart tissue

T. R. Werner et al. / Sci. Rep. / 2019

Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III-secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus

I. Jiménez-Guerrero et al. / Mol. Plant Pathol. / 2019

Allopregnanolone reduces neuroendocrine response to acute stressful stimuli in sheep

T. Misztal et al. / J. Endocrinol. / 2019

Stem Cell Modeling of Neuroferritinopathy Reveals Iron as a Determinant of Senescence and Ferroptosis during Neuronal Aging

A. Cozzi et al. / Stem Cell Reports / 2019

The AMSH3 ESCRT-III-Associated Deubiquitinase Is Essential for Plant Immunity

T. Schultz-Larsen et al. / Cell Rep. / 2018
Storage & Shipping
Storage

Routine storage: -20ºC

Shipping

Shipping and temporary storage for up to 1 month at room temperature has no detrimental effects on the quality of HOT FIREPol® EvaGreen® qPCR Mix Plus.