PerfeCTa SYBR® Green FastMix

Super. Fast. PerfeCTa.

Features & Benefits

  • 2X concentrated master mixes with stabilized SYBR Green dye and exceptional temperature stability (≥30 days at 22°C) that withstand repetitive freeze-thaw (≥ 20X).
  • Rigorously optimized for both fast and conventional thermal cycling parameters.
  • Supports efficient vortex mixing with proprietary anti-foaming technology.
  • Maximizes assay sensitivity and target precision with highly modified Taq DNA polymerase and stringent ultrapure, AccuFast™ II antibody hotstart technology

 

PerfeCTa SYBR Green FastMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Description

PerfeCTa SYBR Green FastMix is a 2X concentrated, ready-to-use reaction cocktail that contains all components, except primers and DNA template. This rigorously optimized master mix contains of proprietary buffer technology, stabilizers and AccuFast Taq DNA polymerase to deliver maximum assay precision, sensitivity, and PCR efficiency for accelerated or conventional thermal cycling conditions for SYBR Green detection. Dye-based detection methods are critically dependent on highly specific amplification because dsDNA dyes will bind to any amplicon, including off-target primer elongation and primer dimerization. AccuFast hot start Taq DNA polymerase contains a proprietary mixture of ultra-pure monoclonal antibodies that stringently suppress primer elongation prior to the initial PCR denaturation step and allows for setup and multi-day storage at ambient room temperature prior to thermal cycling. AccuFast provides rapid release of fully active enzyme to support accelerated thermal cycling conditions.

Performance Data

Comparison to Finnzymes, ADAR


PerfeCTa SYBR Green FastMix Comparison to DyNAmo Flash SYBR Green PCR Kit

RNA-specific adenosine deaminase (ADAR) was amplified from log-fold dilutions of total HeLa cell cDNA (100 ng to 10 pg) using PerfeCTa™ SYBR Green FastMix or the DyNAmo Flash SYBR Green PCR Kit (Finnzymes) according to each manufacturers protocol. Averaged plots for quadruplicate reactions for each input quantity are shown. Fusion of DNA-binding peptide to Tbr DNA pol results in lower specificity of the DyNAmo kit which is evident in false positive results for no template control (NTC) reactions. Chemically modified polymerase produces delayed Cts and lower signal strength compared to AccuStart™ Taq. Cycling conditions: Finnzymes: 95°C, 7 min followed by 40 cycles of 95°C, 10s; 60°C, 20s PerfeCTa™ SYBR Green FastMix: 95°C, 20s followed by 40 cycles of 95°C, 1s; 60°C, 20s


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Comparison to Takara, ADAR


PerfeCTa SYBR Green FastMix Comparison to SYBR PreMix Ex Taq

RNA-specific adenosine deaminase (ADAR) was amplified from log-fold dilutions of total HeLa cell cDNA (100 ng to 10 pg) using PerfeCTa&trade; SYBR Green FastMix or SYBR PreMix Ex Taq&trade; (Takara) according to each manufacturers protocol. Averaged plots for quadruplicate reactions for each input quantity are shown. PerfeCTa&trade; SYBR Green FastMix produces higher fluorescent signal and detection of equal target amounts at earlier Cts. Cycling conditions for both kits: 95&deg;C, 20s followed by 40 cycles of 95&deg;C, 1s; 60&deg;C, 20s


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Comparison to SYBR GreenER, ADAR


PerfeCTa SYBR Green FastMix comparison to SYBR GreenER qPCR SuperMix

RNA-specific adenosine deaminase (ADAR) was amplified from log-fold dilutions of total HeLa cell cDNA (100 ng to 1 pg) using PerfeCTa&trade; SYBR Green FastMix or the SYBR GreenER qPCR SuperMix (Invitrogen) according to each manufacturers protocol. Averaged plots for quadruplicate reactions for each input quantity are shown. Replicate CT values are shown on the standard curve (Panel A, inset). Cycling conditions: Invitrogenn: 95&deg;C, 10 min followed by 40 cycles of 95&deg;C, 10s; 60&deg;C, 60s; PerfeCTa&trade; SYBR Green FastMix: 95&deg;C, 20s followed by 40 cycles of 95&deg;C, 1s; 60&deg;C, 20s. PerfeCTa SYBR Green FastMix amplified the ADAR gene with higher efficiency and greater sensitivity. All replicate reactions for SYBR GreenER qPCR SuperMix failed to amplify ADAR from 1 pg of cDNA.


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Details

  • Contents

    Single-tube, 2X concentrated reagent containing:

    • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, and dTTP.
    • AccuStart II Taq DNA Polymerase
    • SYBR Green I dye
    • Inert AccuVue dye
    • Proprietary enzyme stabilizers and performance-enhancing additives.
    • Titrated reference dye (if applicable)
  • Storage & Handling
    PerfeCTa SYBR Green FastMix is stable for 1 year when stored in a constant temperature freezer at -20°C, protected from light. For convenience, it may be stored unfrozen at 4°C for up to 6 months. After thawing, mix thoroughly before using. Stabilized reagent demonstrates no functional loss of performance after 20 freeze-thaw cycles or 2 months at 20°C.
  • 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
    • BioRad CFX
    • Roche LightCycler 480
    • Other
    Bio-Rad iCycler iQ systems
    • BioRad iCycler iQ™
    • BioRad MyiQ™
    • BioRad iQ™5
  • Related Resources
    Product Manuals
    Product Flyers
    Safety Data Sheets (SDS)
    CofA (PSF)
    Publications
    Topography of microglial activation in sensory- and affect-related brain regions in chronic pain
    Anna M.W. Taylor, Journal of Neuroscience Research - 2016
    Abstract
    Microglial activation in the spinal cord plays a central role in the development and maintenance of chronic pain after a peripheral nerve injury (PNI). There has not yet been a thorough assessment of microglial activation in brain regions associated with pain and reward. To this end, this study uses a mouse model of neuropathic pain in which the left sciatic nerve of male C57Bl/6J mice is loosely constricted (chronic constriction injury) to assess microglial activation in several brain regions 2 weeks after injury, a time point at which pain hypersensitivity is well established. We found significant microglial activation in brain regions associated with sensory pain transmission and affect, including the thalamus, sensory cortex, and amygdala. Activation was consistently most robust in brain regions contralateral to the side of injury. Brain regions not directly involved in either sensory or affective dimensions of pain, such as the motor cortex, did not display microglial activation. This study confirms that PNI induces microglial activation in regions involved with both sensory and affective components of pain. © 2016 Wiley Periodicals, Inc.
    Identification of therapeutic targets for glioblastoma by network analysis
    D. Friedmann-Morvinski, Oncogene - 2015
    Abstract
    Glioblastoma can originate from terminally differentiated astrocytes and neurons- which can dedifferentiate to a stem cell-like state upon transformation. In this study- we confirmed that transformed dedifferentiated astrocytes and neurons acquired a stem/progenitor cell state- although they still retained gene expression memory from their parental cell. Transcriptional network analysis on these cells identified upregulated genes in three main pathways: Wnt signaling- cell cycle and focal adhesion with the gene Spp1- also known as osteopontin (OPN) serving as a key common node connecting these three pathways. Inhibition of OPN blocked the formation of neurospheres- affected the proliferative capacity of transformed neurons and reduced the expression levels of neural stem cell markers. Specific inhibition of OPN in both murine and human glioma tumors prolonged mice survival. We conclude that OPN is an important player in dedifferentiation of cells during tumor formation- hence its inhibition can be a therapeutic target for glioblastoma.
    Expression activation and functional analysis of HLA3- a putative inorganic carbon transporter in Chlamydomonas reinhardtii
    Han Gao, The Plant Journal - 2015
    Abstract
    The CO2 concentrating mechanism (CCM) is a key component of the carbon assimilation strategy of aquatic microalgae. Induced by limiting CO2 and tightly regulated- the CCM enables these microalgae to respond rapidly to varying environmental CO2 supplies and to perform photosynthetic CO2 assimilation in a cost-effective way. A functional CCM in eukaryotic algae requires Rubisco sequestration- rapid interconversion between CO2 and HCO3- catalyzed by carbonic anhydrases (CAs)- and active inorganic carbon (Ci) uptake. In the model microalga Chlamydomonas reinhardtii- a membrane protein HLA3 is proposed to be involved in active Ci uptake across the plasma membrane. In this study- we use an artificially designed transcription activator-like effector (dTALE) to activate the expression of HLA3. The successful activation of HLA3 expression demonstrates dTALE as a promising tool for gene-specific activation and investigation of gene function in Chlamydomonas. Activation of HLA3 expression in high CO2 acclimated cells- where HLA3 is not expressed- resulted in increased Ci accumulation and Ci-dependent photosynthetic O2 evolution specifically in very low CO2 concentrations- which confirms that HLA3 is indeed involved in Ci uptake- and suggests it is mainly associated with HCO3- transport in very low CO2 concentrations- conditions in which active CO2 uptake is highly limited.
    Social context affects immune gene expression in a subterranean termite
    Q. Gao, Insectes Sociaux - 2015
    Abstract
    Social living may burden the immune system by increasing susceptibility to contagion. Animals may- however- evolve socially enabled defenses that limit their demographic vulnerability to disease. In this study we test for evidence of social immunity by exposing single versus grouped termites to a topical application of fungal spores- and measuring the transcriptional response at immune loci. We find that immune-related genes tend to be less responsive within infected groups- compared to the response of singly infected individuals. Two gram-negative binding protein genes (GNBP1- GNBP2) and a leucine-rich repeat immune gene are up-regulated upon infection of singletons- but are not disregulated upon grouped infection. A fourth locus (termicin) was- by contrast- disproportionately up-regulated in social groups- relative to singletons. These socially responsive patterns to immune gene expression in the Eastern subterranean termite suggests a real-time interplay between the individual immune response and the immediate social context.
    Differential induction of plasma cells by isoforms of human TACI
    Yolanda Garcia-Carmona, Blood - 2015
    Abstract
    Subjects with common variable immune deficiency (CVID) may have mutations in trans-membrane activator calcium modulator and cyclophylin ligand interactor (TACI). Unlike the murine gene- human TACI undergoes alternative mRNA splicing to produce isoforms with one or two ligand-binding domains. Since both are found in human B cells- we compared their functions in transduced murine B and human pre-B cells. While murine cells and even more dramatically- pre-B cells transduced with the long TACI isoform- retained surface CD19 and IgG- cells transduced with the TACI short isoform completely lost these B cell characteristics. Expression of the short TACI isoform produced intense NF-kB activation- nuclear p65 translocation and colocalization with MyD88 and CAML. The short TACI transduced cells became larger- CD138+ with upregulated BLIMP1 and XBP1 mRNA- and acquired the morphology of plasma cells. In contrast- cells bearing the long isoform had significantly less BLIMP1 and XBP1 mRNA and for human pre-B cells- remained CD138neg. While human B cells express both isoforms- the short isoform predominates in CD27+ B cells- TLR9-activated peripheral B cells and splenic marginal zone B cells. While the transcriptional controls for alternative splicing of isoforms remain unknown- differential signals via isoforms may control plasma cell generation in humans.
    Click here to see all Publications
    FAQs
    Can I use PerfeCTa® SYBR® Green FastMix® instead of SYBR Green SuperMix?
    You can use FastMix instead of SuperMix. We have observed similar results with both PCR mixes, however, slightly lower background was observed using SuperMix when using 10 ng of cDNA or more in the qPCR. The qScript microRNA Quantification system has been validated using SYBR Green SuperMix.
    Click here to see all FAQs

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