AccuStart Genotyping ToughMix

enables probe-based genetic analysis (SNP detection and allelic discrimination) directly from crude extracts, DBS punches, plant tissue and clinical specimens

Features & Benefits

  • Optimized buffer chemistry destabilizes single base-pair mismatch probes, providing superior allelic discrimination and improved cluster separation for critical, single-nucleotide polymorphism (SNP) detection assays
  • Sensitive, precise detection with ultrapure AccuStart II enzyme technology – maximum-yielding Taq DNA polymerase mutant controlled by stringent, multi-epitope antibody hotstart
  • ToughMix reagent technology neutralizes a broad spectrum of PCR inhibitors common in plant and animal tissues, environmental sources, clinical specimens and complex food matrices
  • Easy-to-use 2x concentrated SuperMix with AccuVue plate loading dye and pre-blended passive reference dye simplifies reaction setup
  • Supports efficient vortex mixing with proprietary anti-foaming technology


Genotyping ToughMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.


Genotyping ToughMix is a 1-tube qPCR SuperMix reagent compatible with all dual-label (hydrolysis) probe chemistries for both fast and conventional PCR cycling protocols or instruments. This proprietary formulation has been rigorously optimized to destabilize single base-pair mismatches to ensure precise allelic discrimination and cluster separation with SNP detection assays. The reagent is provided as a 2x concentrated readyto- use reaction cocktail that contains all required reaction components, except primers, probe(s), and DNA template. Inert AccuVue plate loading dye helps to minimize pipette error and provides visual confirmation of thorough mixing.

Performance Data

ToughMix vs Competitor

Comparison to conventional master mixes

AccuStart Genotyping ToughMix stands up to the challenge where other genotyping master mixes fall apart. ToughMix can be used with clean templates where it generates higher fluorescent signal and tighter clusters than the leading competitors mix.


Influence of PCR inhibitor

Comparison to conventional master mixes

In the presence of a common PCR inhibitor, humic acid (50ng/l), the competitors system is completely shut down while ToughMix delivers robust, accurate results



  • Contents

    Single-tube, 2X concentrated reagent containing:

    • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP.
    • AccuStart II Taq DNA Polymerase.
    • Inert AccuVue dye.
    • Proprietary enzyme stabilizers and performance-enhancing additives.
    • Titrated reference dye (if applicable).
  • Storage & Handling
    Store components in a constant temperature freezer at -25°C to -15°C protected from light upon receipt. After thawing, mix thoroughly before using. Repeated freezing and thawing does not affect PCR performance. For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.
  • Instrument Capability
    • Applied Biosystems 5700
    • Applied Biosystems 7000
    • Applied Biosystems 7300
    • Applied Biosystems 7700
    • Applied Biosystems 7900
    • Applied Biosystems 7900HT
    • Applied Biosystems 7900 HT Fast
    • Applied Biosystems StepOne™
    • Applied Biosystems StepOnePlus™
    Low ROX
    • Applied Biosystems 7500
    • Applied Biosystems 7500 Fast
    • Stratagene Mx3000P®
    • Stratagene Mx3005P™
    • Stratagene Mx4000™
    • Applied Biosystems ViiA 7
    • Applied Biosystems QuantStudio™
    • Agilent AriaMx
    • Douglas Scientific IntelliQube®
    No ROX
    • Quantabio Q
    • BioRad CFX
    • Roche LightCycler 480
    • QIAGEN Rotor-Gene Q
    • Other
    Bio-Rad iCycler iQ systems
    • BioRad iCycler iQ™
    • BioRad MyiQ™
    • BioRad iQ™5
  • Related Resources
    Product Flyers
    Product Manuals
    A functional genetic marker for apple red skin coloration across different environments
    David Chagné, Tree Genetics & Genomes - 2016
    The red skin color desired by most apple consumers is not easy to achieve in warm climates, as the expression of MYB10, which regulates red pigmentation in apple, is influenced negatively by high temperatures. We describe the development and validation of a genetic marker for red skin coloration that effectively predicts color in a warm summer environment in Spain, as well as more temperate climates in New Zealand and Italy. Following the determination of a major-effect quantitative trait locus (QTL) controlling red skin coloration on linkage group (LG)9, using four segregating populations grown in New Zealand, and screened using the IRSC apple 8-K single-nucleotide polymorphism (SNP) array, the most significant SNP marker (ss475879531) was transformed into a marker suitable for use in a real-time PCR assay. This marker was validated using five apple seedling populations growing in a warm summer environment in Spain, demonstrating that the marker system efficiently predicts red skin coloration and can be used for marker assisted selection, even under conditions considered adverse for skin color development.
    Based Approach for Investigating Cardiovascular Disease-Related Genetic Variants
    Sukhvir K. Rai BSc, MSc, University of Leicester - 2016
    Genome wide association studies (GWASs) have identified many loci that contribute to coronary artery disease (CAD). A huge challenge of the post GWAS era is identifying the biological pathways risk variants act through to contribute to disease. I explored the use of MSCs as a model to investigate the effects of CAD related variants in vitro. To establish a MSC bank, cells were isolated from 114 umbilical cords and subsequently characterised. MSCs met the ISCT criteria; they adhered to plastic, expressed a characteristic cell surface profile and differentiated into three common lineages. As a proof of principle study, a BMI ssociated variant was investigated. Speliotes et l., 2010) showed rs3810291 was an eQTL for ZC3H4 in adipose tissue. However, MSC erived adipocytes did not recapitulate the effect of this variant. Analysis of another known eQTL (rs10840106) in adipose tissue was significantly associated with TRIM66 ene expression in MSCs and differentiated adipocytes. It is hypothesised CAD risk variants act in CAD relevant cell types to exert their effects, so MSCs were differentiated towards a SMC lineage. A second proof of concept study focussed on a CAD associated variant reported to be functional in SMCs. Puet al., (2013) showed rs3825807 affected vascular SMC migration, ADAMTS7 maturation and COMP cleavage. I did not see a genotype effect of rs3825807 on ADAMTS7 prodomain cleavage or migration of differentiated SMCs. These findings suggest that MSCs may be robust enough to detect the association of variants on important genetic effects such as mRNA levels. However using MSCs to understand the effects of CAD -associated variants is still a difficult process. It is hindered by technical challenges such as MSC heterogeneity, variation in MSC differentiation and underpowered sample sizes. These barriers need to be overcome in order to successfully use MSCs to model disease.
    Geula Hanin, Human Molecular Genetics - 2014
    MicroRNAs (miRNAs) can repress multiple targets- but how a single de-balanced interaction affects others remained unclear. We found that changing a single miRNA-target interaction can simultaneously affect multiple other miRNA-target interactions and modify physiological phenotype. We show that miR-608 targets acetylcholinesterase (AChE) and demonstrate weakened miR-608 interaction with the rs17228616 AChE allele having a single-nucleotide polymorphism (SNP) in the 3'-untranslated region (3'UTR). In cultured cells- this weakened interaction potentiated miR-608-mediated suppression of other targets- including CDC42 and interleukin-6 (IL6). Postmortem human cortices homozygote for the minor rs17228616 allele showed AChE elevation and CDC42/IL6 decreases compared with major allele homozygotes. Additionally- minor allele heterozygote and homozygote subjects showed reduced cortisol and elevated blood pressure- predicting risk of anxiety and hypertension. Parallel suppression of the conserved brain CDC42 activity by intracerebroventricular ML141 injection caused acute anxiety in mice. We demonstrate that SNPs in miRNA-binding regions could cause expanded downstream effects changing important biological pathways.
    Obesity, diabetes, and leptin resistance promote tau pathology in a mouse model of disease - ClinicalKey
    T.L. Platt, Neuroscience - 2016
    Obesity and type 2 diabetes mellitus (T2DM) convey an increased risk for developing dementia. The microtubule-associated protein tau is implicated in neurodegenerative disease by undergoing hyperphosphorylation and aggregation, leading to cytotoxicity and neurodegeneration. Enzymes involved in the regulation of tau phosphorylation, such as GSK3β, are tightly associated with pathways found to be dysregulated in T2DM. We have shown previously that leptin-resistant mice, which develop obesity and a diabetic phenotype, display elevated levels of tau phosphorylation. Here we show cells cultured with leptin, an adipokine shown to have neuroprotective effects, reduces tau phosphorylation. To explore how this mechanism works in vivo we transduced an existing diabetic mouse line (Leprdb/db) with a tau mutant (tauP301L) via adeno-associated virus (AAV). The resulting phenotype included a striking increase in tau phosphorylation and the number of neurofibrillary tangles (NFTs) found within the hippocampus. We conclude that leptin resistance-induced obesity and diabetes accelerates the development of tau pathology. This model of metabolic dysfunction and tauopathy provides a new system in which to explore the mechanisms underlying the ways in which leptin resistance and diabetes influence development of tau pathology, and may ultimately be related to the development of NFTs.
    Click here to see all Publications
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    CofA (PSF)

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