5PRIME HotMasterMix

Features & Benefits

  • Optimal results – highly specific amplification
  • Minimal handling – ready-to-use mastermix format
  • Convenience – storage at 4°C eliminates freeze-thaw cycles

 

5PRIME HotMasterMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Description

The 5PRIME HotMasterMix is a ready-to-use reagent mix that offers high reproducibility when processing large numbers of samples. The HotMaster Taq DNA Polymerase, an integral component of the master mix, is designed to reduce or eliminate any non-specific products that may result from mispriming during PCR.

The 5PRIME HotMasterMix (2.5x) is a ready-to-use PCR mix. Only primers and template need to be added to the 2.5x concentrate, thus decreasing the number of time-consuming pipetting steps. This format not only reduces the likelihood of errors and the risk of contamination, but it also increases precision and sample throughput.

The 5PRIME HotMasterMix also contains the self-adjusting Mg2+ buffer technology. This formulation adjusts the Mg2+ concentration automatically, eliminating the need for optimizing this critical component. The MasterMix does not need to be stored frozen, eliminating the time-consuming thawing process and the resulting reduction in performance.

The 5PRIME HotMasterMix is a 2.5-fold concentrate, resultingin the following final concentrations for a 50 μl PCR reaction: 1 U Taq DNA Polymerase, 45 mM Cl, 2.5 mM Mg2+, 200 μM of each dNTP.

Details

  • Contents
    2.5x HotMasterMix solution
  • Storage & Handling
    Store components in a constant temperature freezer at -25°C to -15°C upon receipt. For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.
    Once frozen, HotMasterMix must be completely thawed and mixed well before returning to -20°. Never thaw and re-freeze without mixing
  • Related Resources
    CofA (PSF)
    Product Manuals
    Safety Data Sheets (SDS)
    Publications
    Effect of freshwater mussels on the vertical distribution of anaerobic ammonia oxidizers and other nitrogen-transforming microorganisms in upper Mississippi river sediment [PeerJ]
    Ellen M. Black, PeerJ - 2017
    Abstract
    Targeted qPCR and non-targeted amplicon sequencing of 16S rRNA genes within sediment layers identified the anaerobic ammonium oxidation (anammox) niche and characterized microbial community changes attributable to freshwater mussels. Anammox bacteria were normally distributed (Shapiro-Wilk normality test, W-statistic =0.954, p = 0.773) between 1 and 15 cm depth and were increased by a factor of 2.2 (p < 0.001) at 3 cm below the water-sediment interface when mussels were present. Amplicon sequencing of sediment at depths relevant to mussel burrowing (3 and 5 cm) showed that mussel presence reduced observed species richness (p = 0.005), Chao1 diversity (p = 0.005), and Shannon diversity (p < 0.001), with more pronounced decreases at 5 cm depth. A non-metric, multidimensional scaling model showed that intersample microbial species diversity varied as a function of mussel presence, indicating that sediment below mussels harbored distinct microbial communities. Mussel presence corresponded with a 4-fold decrease in a majority of operational taxonomic units (OTUs) classified in the phyla Gemmatimonadetes, Actinobacteria, Acidobacteria, Plantomycetes, Chloroflexi, Firmicutes, Crenarcheota, and Verrucomicrobia. 38 OTUs in the phylum Nitrospirae were differentially abundant (p < 0.001) with mussels, resulting in an overall increase from 25% to 35%. Nitrogen (N)-cycle OTUs significantly impacted by mussels belonged to anammmox genus Candidatus Brocadia, ammonium oxidizing bacteria family Nitrosomonadaceae, ammonium oxidizing archaea genus Candidatus Nitrososphaera, nitrite oxidizing bacteria in genus Nitrospira, and nitrate- and nitrite-dependent anaerobic methane oxidizing organisms in the archaeal family “ANME-2d” and bacterial phylum “NC10”, respectively. Nitrosomonadaceae (0.9-fold (p < 0.001)) increased with mussels, while NC10 (2.1-fold (p < 0.001)), ANME-2d (1.8-fold (p < 0.001)), and Candidatus Nitrososphaera (1.5-fold (p < 0.001)) decreased with mussels. Co-occurrence of 2-fold increases in Candidatus Brocadia and Nitrospira in shallow sediments suggests that mussels may enhance microbial niches at the interface of oxic–anoxic conditions, presumably through biodeposition and burrowing. Furthermore, it is likely that the niches of Candidatus Nitrososphaera and nitrite- and nitrate-dependent anaerobic methane oxidizers were suppressed by mussel biodeposition and sediment aeration, as these phylotypes require low ammonium concentrations and anoxic conditions, respectively. As far as we know, this is the first study to characterize freshwater mussel impacts on microbial diversity and the vertical distribution of N-cycle microorganisms in upper Mississippi river sediment. These findings advance our understanding of ecosystem services provided by mussels and their impact on aquatic biogeochemical N-cycling.
    Pinpointing recurrent proviral integration sites in new models for latent HIV-1 infection
    Ulrike C. Lange, Virus Research - 2018
    Abstract
    HIV infection is characterized by accumulation of proviral sequences within the human host genome. Integration of viral-derived DNA occurs at preferential loci, suggesting a site-specific crosstalk between viral sequences and human genes. We here describe a genome engineering workflow to generate models for HIV-1 infection that for the first time recapitulate proviral integration at selected genomic loci and provide unique tools to study effects of HIV proviral integration site choice. Using this workflow, we have derived two BACH2–HIV-1 reporter models that mimic largely latent integration in the clinically relevant BACH2 gene locus, which has been associated with recurrent integration and HIV-reservoir maintenance in chronically infected patients.
    Characterization of benthic biogeochemistry and ecology at three methane seep sites on the Northern U.S. Atlantic margin
    D. McVeigh, Deep Sea Research Part II: Topical Studies in Oceanography - 2018
    Abstract
    Several hundred methane seeps were recently discovered along the U.S. Atlantic margin, a passive margin without a major hydrocarbon basin. These seeps represent a compelling opportunity to compare and contrast geochemical parameters, as well as the distribution and composition of associated chemosynthetic ecosystems, across geologically distinct seeps. Specifically, this study characterized the physical, chemical, geological, and biological features at Veatch Canyon, New England, and Shallop Canyon methane seep sites using a suite of state-of-the-art sampling and communication tools: AUV Sentry, HOV Alvin, a video-equipped multicore, and a real-time telepresence connection to the Inner Space Center (ISC) at the University of Rhode Island. Water column backscatter data collected by AUV Sentry confirmed previously detected gas emission and further indicated the presence of 13 actively emitting gas seeps within the Veatch Canyon survey area, 5 within the New England survey area, and 15 within the Shallop Canyon survey area. Complementary high-resolution seafloor bathymetry and backscatter mapping data indicated highly rugose morphology at all surveyed seep sites including the presence of extensive rock outcrops and bivalve beds. Video collected by HOV Alvin provided visual confirmation of gas plumes and the presence of chemosynthetic communities, including microbial mats and mussel beds, as well as large outcrops of authigenic carbonate rock surrounding locations of discrete gas emission. Bathymodiolus was the dominant species observed at the Veatch Canyon methane seep site, while the deep-sea red crab, Chaceon quinquedens, dominated the New England and Shallop Canyon seep sites. Elevated pore water sulfide concentrations suggested stimulation of sulfate-coupled anaerobic oxidation of methane (AOM) and/or degradation of organic matter in seep sediments relative to non-seep sediments; AOM was observed directly within overlying authigenic carbonate rocks. DNA sequencing of the surface sediments revealed diverse sulfide-oxidizing bacteria similar to the taxa described previously at other methane seeps, including members of the Thiotrichales and Campylobacterales. Together, these analyses provide one of the first interdisciplinary descriptions of sites within this massive seep system, setting the stage for more targeted, hypothesis-driven investigations. In this context, telepresence facilitated interdisciplinary research between specialists at the ISC and scientists and engineers at sea, enabling researchers to transfer data and maximize research efforts with sampling tools at sea. Our findings suggest that although the geological setting is distinct, the surficial microbial and macrofaunal communities closely resemble those of previously described methane seeps.
    A Method for Targeted 16S Sequencing of Human Milk Samples
    Tobin, N. H., J. Vis. Exp. - 2018
    Abstract
    Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.
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    Product
    Kit Size
    Order Info
  • 5PRIME HotMasterMix
    100 x 50 μL rxns (2 x 1 mL)
  • 5PRIME HotMasterMix
    1000 x 50 μL rxns (20 x 1 mL)