PerfeCTa qPCR FastMix UNG

Features & Benefits

  • Fast Cycling protocols
  • contains UNG to eliminate amplification of carry-over contamination

PerfeCTa qPCR FastMix UNG Kits are intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Description

PerfeCTa qPCR FastMix, UNG is a 2X concentrated, ready-to-use reaction cocktail that contains all components, except primers, probe(s), and template for real-time quantitative PCR systems that do not require an internal reference dye. The proprietary buffer and stabilizers have been specifically optimized to deliver maximum PCR efficiency, sensitivity, and robust fluorescent signal with TaqMan® or TaqMan MGB probe chemistry when using rapid PCR cycle times and reduced reaction volumes. This affords greater reagent economy and laboratory throughput on conventional or rapid ramp rate qPCR systems. The enhanced specificity of this FastMix suppresses cross-reactivity between homologous sequences, improving detection and discrimination in SNP applications. A key component of this FastMix is AccuFast™ Taq DNA polymerase. This hot-start Taq contains a proprietary mixture of monoclonal antibodies that bind to the polymerase and keep it inactive prior to the initial PCR denaturation step (> 48 hours at room temperature). Similar to our AccuStart™ Taq DNA polymerase, these antibodies are irreversibly inactivated during the initial PCR denaturation step. However, unlike other antibody hot-start polymerases, activation of AccuFast Taq is instantaneous at 95ºC. Rapid recovery of fully active, unmodified Taq DNA polymerase is critical for efficient extension kinetics. Replication of fragments up to 200 bp is complete in less than 20s at 60ºC. Additionally, the dNTP mix in this FastMix contains dUTP in place of dTTP. Inclusion of uracil-N-glycosylase (UNG) prevents amplification of carry-over contamination from previous dU-containing PCRs.

Details

  • Contents
    2X reaction buffer containing optimized concentrations of MgCl2, dNTPs, AccuFast Taq DNA Polymerase, UNG, and stabilizers
  • Storage & Handling
    Store components in a constant temperature freezer at -25°C to -15°C protected from light upon receipt. For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.
  • Instrument Capability
    ROX
    • 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®
    • QIAGEN Rotor-Gene Q
    No ROX
    • Quantabio Q
    • BioRad CFX
    • Roche LightCycler 480
    • Other
    Bio-Rad iCycler iQ systems
    • BioRad iCycler iQ™
    • BioRad MyiQ™
    • BioRad iQ™5
  • Related Resources
    Product Manuals
    Safety Data Sheets (SDS)
    CofA (PSF)
    Publications
    N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis
    Thulasi Warrier, Proceedings of the National Academy of Sciences - 2016
    Abstract
    DNA from dust: comparative genomics of large DNA viruses in field surveillance samples - 5ea6c536d204e515562491e37d3dacaa8380.pdf
    Utsav Pandey, Pennsylvania State University - 2016
    Abstract
    The intensification of the poultry industry over the last sixty years facilitated the evolution of increased virulence and vaccine breaks in Marek’s disease virus (MDV-1). Full genome sequences are essential for understanding why and how this evolution occurred, but what is known about genome-wide variation in MDV comes from laboratory culture. To rectify this, we developed methods for obtaining high quality genome sequences directly from field samples without the need for sequence-based enrichment strategies prior to sequencing. We applied this to the first characterization of MDV-1 genomes from the field, without prior culture. These viruses were collected from vaccinated hosts that acquired naturally circulating field strains of MDV-1, in the absence of a disease outbreak. This reflects the current issue afflicting the poultry industry, where virulent field strains continue to circulate despite vaccination, and can remain undetected due to the lack of overt disease symptoms. We found that viral genomes from adjacent field sites had high levels of overall DNA identity, and despite strong evidence of purifying selection, had coding variations in proteins associated with virulence and manipulation of host immunity. Our methods empower ecological field surveillance, make it possible to determine the basis of viral virulence and vaccine breaks, and can be used to obtain full genomes from clinical samples of other large DNA viruses, known and unknown.
    Distinct activation mechanisms trigger the trypanocidal activity of DNA damaging prodrugs
    Emma Louise Meredith, Molecular Microbiology - 2017
    Abstract
    Quinone-based compounds have been exploited to treat infectious diseases and cancer, with such chemicals often functioning as inhibitors of key metabolic pathways or as prodrugs. Here, we screened an aziridinyl-1,4-benzoquinone (ABQ) library against the causative agents of trypanosomiasis, and cutaneous leishmaniasis, identifying several potent structures that exhibited EC50 values of <100 nM. However, these compounds also displayed significant toxicity towards mammalian cells indicating that they are not suitable therapies for systemic infections. Using anti-T. brucei ABQs as chemical probes, we demonstrated that these exhibit different trypanocidal modes of action. Many functioned as type I nitroreductase (TbNTR) or cytochrome P450 reductase (TbCPR) dependent prodrugs that, following activation, generate metabolites which promote DNA damage, specifically interstrand crosslinks (ICLs). Trypanosomes lacking TbSNM1, a nuclease that specifically repairs ICLs, are hypersensitive to most ABQ prodrugs, a phenotype exacerbated in cells also engineered to express elevated levels of TbNTR or TbCPR. In contrast, ABQs that contain substituent groups on the biologically active aziridine do not function as TbNTR or TbCPR-activated prodrugs and do not promote DNA damage. By unravelling how ABQs mediate their activities, features that facilitate the desired anti-parasitic growth inhibitory effects could be incorporated into new, safer compounds targeting these neglected tropical diseases. This article is protected by copyright. All rights reserved.
    Mucosa-associated lymphoid tissue lymphoma translocation 1 as a novel therapeutic target for rheumatoid arthritis
    Chang Hoon Lee, Scientific Reports - 2017
    Abstract
    Emerging evidence suggests that mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) is a key regulator of inflammatory diseases; however, the pathological role of MALT1 in rheumatoid arthritis (RA) is not well understood. Consequently, this protein has not been therapeutically targeted for the treatment of RA. MALT1 plays a role in the paracaspase pathway, has proteolytic activity and is involved in the regulation of inflammatory responses. In this study, we found that the MALT1-targeting inhibitory small molecule, MALT1 selective inhibitor 2-chloro-N-[4-[5-(3,4-dichlorophenyl)-3-(2-methoxyethoxy)-1H-1,2,4-triazol-1-yl]phenylacetamide (MI-2) strongly suppresses the differentiation of monocytes into osteoclasts in the absence or presence of the inflammatory cytokine tumour necrosis factor α. Furthermore, MI-2 ameliorates pathologic bone erosion and synovitis in an in vivo mouse model of collagen-induced arthritis. Mechanistically, MI-2 blocked expression of the master osteoclast regulator – nuclear factor of activated T cells 1 (NFATc1) – by inhibiting nuclear factor κB (NF-κB), which is a critical regulator of NFATc1. These findings highlight the important regulatory role of MALT1 in the NF-κB–NFATc1-signalling axis during osteoclastogenesis and suggest that targeting MALT1 is a promising treatment option for rheumatoid arthritis.
    Iron oxide nanoparticles enhance Toll-like receptor-induced cytokines in a particle size- and actin-dependent manner in human blood
    Susann Wolf-Grosse, Nanomedicine - 2018
    Abstract
    Aim: To assess the effects of different-sized iron oxide nanoparticles (IONPs) on inflammatory responses in human whole blood. Materials & methods: Human whole blood with and without 10 and 30 nm IONPs was incubated with Toll-like receptor (TLR) ligands. Cytokine levels, complement activation, reactive oxygen species and viability were determined. Results: The 10 nm IONPs enhanced the TLR2/6, TLR4 and partly TLR8-mediated cytokine production, whereas the 30 nm IONPs partly enhanced TLR2/6 and decreased TLR8-mediated cytokine production. Particle-mediated enhancement of TLR4-induced cytokines could not be explained by complement activation, but was dependent on TLR4/MD2 and CD14, as well as actin polymerization. Conclusion: The IONPs differentially affected the TLR ligand-induced cytokines, which has important implications for biomedical applications of IONPs.
    Click here to see all Publications
    FAQs
    How stable is an experiment set up with the PerfeCTa qPCR Fastmix Kit at room temperature?
    For optimal results, it is recommended to directly run the reaction plate after setting up the reaction. If for any reason a reaction plate cannot be run within 2 hours of completing the reaction setup, then freeze/refrigerate the reaction plate until it can be loaded and run on a Real-Time PCR System.
    What are the applications for the PerfeCTa qPCR FastMix?
    Absolute Quantitation and Relative Quantitation assays can be run fast. Allelic Discrimination (SNP) assays and plus/minus assays using internal positive controls cannot be run fast.
    How do assays performed with PerfeCTa qPCR FastMix compare with assays performed with PerfeCTa qPCR FastMix?
    In general, assays have been found to perform comparably. Please consult the PerfeCTa qPCR Supermix (2X) Protocol for more information on using this reagent.
    What component in the PerfeCTa qPCR FastMix allows rapid amplification results?
    The hot-start DNA polymerase enzyme system in the PerfeCTa qPCR FastMix only requires a 30 seconds activation step, and supports significantly shorter hold times for the thermal cycling stages of PCR, thereby minimizing PCR run time. For more information on using this reagent, please consult the PerfeCTa qPCR FastMix (2X) protocol.
    SNP assays with PerfeCTa qPCR FastMix?
    The TaqMan® SNP Genotyping assays have not been fully tested on the Applied Biosystems 7500 Fast System.
    Click here to see all FAQs