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repliQa HiFi ToughMix

Superior speed and inhibitor tolerance
Features & Benefits
  • High Fidelity – >90x wild type Taq
  • Extreme Speed – up to 3x faster PCR results with extension rates as fast as 1 kb/sec*
  • Tough Tested – tolerant to a wide range of PCR inhibitors
  • Superior Sensitivity – higher yields from lower inputs as little as 2 pg
  • Long Range – amplify +24 kb gDNA, +40 kb λDNA

 

*for fragments less than 1 kb in size

 

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

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repliQa HiFi ToughMix

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Kit Size:  25 rxns
Part Number:  95200-025
Price:  $54.00
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Kit Size:  100 rxns
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Kit Size:  500 rxns
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Description

The repliQa HiFi ToughMix is a 2x, ready-to-use master mix that contains all the components for high fidelity PCR amplification, including a genetically modified DNA polymerase coupled with hot start antibodies. This unique, next generation master mix provides 90x higher fidelity compared to Taq, while reducing time to PCR results by 2-3x. The extreme speed is enabled by extension times as fast as 1-10 kb/sec depending on target length. The enzyme is coupled with the industry leading ToughMix which is tolerant to a wide variety of inhibitors making it suitable for routine PCR, cloning, amplicon sequencing and site directed mutagenesis.

Customer Testimonial

I used NEB Q5 for the same plasmid for PCR, same annealing temperature. Only the repliQa HiFi toughMix gave me the correct size products. It is very fast and works much better than NEB Q5 for difficult plasmids in PCR.

Dr. Kara C.
UT Southwestern Medical Center

Details

Details

Contents

2x reaction buffer containing optimized concentrations of MgCl2, dNTP’s and proprietarily formulated HiFi polymerase, hot start antibodies and ToughMix chemistry

Customer Testimonials

Customer Testimonials

repliQa HiFi ToughMix
"Worked well with our 16s amplicon set up. Clean products, no extraneous peaks."
Next Generation Genomics Specialist | Virginia Tech
repliQa HiFi ToughMix

"Generated 625 bp fragment w/o errors (sequence-confirmed) using 3-step protocol. Worked ~3 times faster than conventional method"

Professor | Vanderbilt University
repliQa HiFi ToughMix

" After struggling to amplify a roughly 5 kb fragment, I tested the repliQa HiFi ToughMix on the same template using a two step PCR per the suggested protocol. It amplified on the first try. Perhaps the most impressive part was the speed. With the suggested extension time of 5 seconds per kb, total reaction time was reduced by two hours compared to the previous polymerase. Subsequent cloning and sequencing showed the amplified fragment contained zero errors. I was quite pleased with this product. "

Gary G. | Scientist, OMRF
repliQa HiFi ToughMix

"All I can say from testing repliQa HiFi ToughMix is that the results are jaw-dropping. The Platinum™ SuperFi II PCR Master Mix did nothing in the same amount of time under the same conditions. The assay I was testing out is a new way to amplify entire mitogenomes as a single amplicon (~15kb). This is a game changer, cutting the PCR time from ~8.5hrs to 1.5hrs, and way more product is generated. I am not often floored by data, but these results are amazing. I have another project I am working on with this assay and I am going to switch to this polymerase for sure. Good work Quantabio!"

A. K. | Post Doc, BCM
repliQa HiFi ToughMix

"The kit have a great power of amplification and extreme fast speed, I just need one hour to get result which I usually spent three hours before."

Simon S. | Professor, Tennessee State University
repliQa HiFi ToughMix

"This can amplify the tough template for SURE. I was struggled with the tough template that showed poor quality results with other many PCR enzymes. This repliQa saved my life and eventually gave me the clear and strong band by gel."

Lili C. | Post Doc, USC
repliQa HiFi ToughMix

"I used NEB Q5 for the same plasmid for PCR, same annealing temperature. Only the repliQa HiFi toughMix gave me the correct size products. It is very fast and works much better than NEB Q5 for difficult plasmids in PCR."

Dr. Kara C. | UT Southwestern Medical Center
repliQa HiFi ToughMix

"Worked great amplifying two gene target from plasmid DNA. It was the first time I have performed a two step reaction and it worked great. It was awesome running everything in 23 minutes too."

Dr. Brandon L. | University of Nebraska

Details

Contents

2x reaction buffer containing optimized concentrations of MgCl2, dNTP’s and proprietarily formulated HiFi polymerase, hot start antibodies and ToughMix chemistry

Performance Data

Resources

Flyers

Product Manuals

Application Notes

CofA (PSFs)

SDSs

Publications

Whole Genome Sequencing of SARS-CoV-2 for Canada’s COVID19 Genomic Surveillance
Grace Eunchong Seo - 2023
Abstract
At the onset of the COVID-19 pandemic, researchers around the globe joined forces to study the evolutionary biology of SARS-CoV-2 and identify approaches to minimize its spread in the population. Whole genome sequencing (WGS) quickly became the gold standard for monitoring SARS-CoV-2 viral evolution and how it may impact disease severity, transmission, and vaccine efficacy. As a result, researchers demonstrated concerted efforts to develop, optimize, and validate methods for WGS of the novel virus. This research herein optimized wet-lab sequencing protocols developed by the international research group using reverse transcription PCR-tiling and nanopore sequencing technologies to sequence the whole genome of SARS-CoV-2. As a part of the Canadian COVID Genomics Network (CanCOGeN), the work presented in this thesis outlines materials, methods, and results that contributed to the optimization and validation of a Canadian-specific SARS-CoV-2 WGS approach. To achieve this goal, the investigation included identifying the most economical reverse transcriptase, DNA polymerase, PCR primer schemes, library preparation conditions, and comparison of Nanopore and Illumina sequencing platforms. The optimized WGS protocol was shared with the CanCOGeN partners across Canada to increase Canada’s SARS-CoV-2 sequencing capacity towards a sustainable national genomic surveillance program. Throughout this project, the WGS protocol was validated to ensure that emerging variants of concern were detectable. In summary, the current research contributed to operationalizing the first national genomic surveillance program for viral pathogens in Canada. The developed protocol remains in use across Canadian partners and contributes data to support evidence-based decisions for implementing pub- lic health measures. Thanks to our work through the CanCOGeN project and its network of expertise, Canada is well-positioned and prepared to perform genomic surveillance of emerging and reemerging pathogens should a new public health threat arise.
Programmable de novo designed coiled coil-mediated phase separation in mammalian cells
Maruša Ramšak - 2023
Abstract
Membraneless liquid compartments based on phase-separating biopolymers have been observed in diverse cell types and attributed to weak multivalent interactions predominantly based on intrinsically disordered domains. The design of liquid-liquid phase separated (LLPS) condensates based on de novo designed tunable modules that interact in a well-understood, controllable manner could improve our understanding of this phenomenon and enable the introduction of new features. Here we report the construction of CC-LLPS in mammalian cells, based on designed coiled-coil (CC) dimer-forming modules, where the stability of CC pairs, their number, linkers, and sequential arrangement govern the transition between diffuse, liquid and immobile condensates and are corroborated by coarse-grained molecular simulations. Through modular design, we achieve multiple coexisting condensates, chemical regulation of LLPS, condensate fusion, formation from either one or two polypeptide components or LLPS regulation by a third polypeptide chain. These findings provide further insights into the principles underlying LLPS formation and a design platform for controlling biological processes.
Programmable de novo designed coiled coil-mediated phase separation in mammalian cells
Maruša Ramšak - 2023
Abstract
Membraneless liquid compartments based on phase-separating biopolymers have been observed in diverse cell types and attributed to weak multivalent interactions predominantly based on intrinsically disordered domains. The design of liquid-liquid phase separated (LLPS) condensates based on de novo designed tunable modules that interact in a well-understood, controllable manner could improve our understanding of this phenomenon and enable the introduction of new features. Here we report the construction of CC-LLPS in mammalian cells, based on designed coiled-coil (CC) dimer-forming modules, where the stability of CC pairs, their number, linkers, and sequential arrangement govern the transition between diffuse, liquid and immobile condensates and are corroborated by coarse-grained molecular simulations. Through modular design, we achieve multiple coexisting condensates, chemical regulation of LLPS, condensate fusion, formation from either one or two polypeptide components or LLPS regulation by a third polypeptide chain. These findings provide further insights into the principles underlying LLPS formation and a design platform for controlling biological processes.
Abundant Sulfitobacter marine bacteria protect Emiliania huxleyi algae from pathogenic bacteria
Roni Beiralas - 2023
Abstract
Emiliania huxleyi is a unicellular micro-alga that forms massive oceanic blooms and plays key roles in global biogeochemical cycles. Mounting studies demonstrate various stimulatory and inhibitory influences that bacteria have on the E. huxleyi physiology. To investigate these algal-bacterial interactions, laboratory co-cultures have been established by us and by others. Owing to these co-cultures, various mechanisms of algal-bacterial interactions have been revealed, many involving bacterial pathogenicity towards algae. However, co-cultures represent a significantly simplified system, lacking the complexity of bacterial communities. In order to investigate bacterial pathogenicity within an ecologically relevant context, it becomes imperative to enhance the microbial complexity of co-culture setups. Phaeobacter inhibens bacteria are known pathogens that cause the death of E. huxleyi algae in laboratory co-culture systems. The bacteria depend on algal exudates for growth, but when algae senesce, bacteria switch to a pathogenic state and induce algal death. Here we investigate whether P. inhibens bacteria can induce algal death in the presence of a complex bacterial community. We show that an E. huxleyi-associated bacterial community protects the alga from the pathogen, although the pathogen occurs within the community. To study how the bacterial community regulates pathogenicity, we reduced the complex bacterial community to a five-member synthetic community (syncom). The syncom is comprised of a single algal host and five isolated bacterial species, which represent major bacterial groups that are naturally associated with E. huxleyi. We discovered that a single bacterial species in the reduced community, Sulfitobacter pontiacus, protects the alga from the pathogen. We further found that algal protection from P. inhibens pathogenicity is a shared trait among several Sulfitobacter species. Algal protection by bacteria might be a common phenomenon with ecological significance, which is overlooked in reduced co-culture systems.
Vaginal microbiome community state types and high-risk human papillomaviruses in cervical precancer and cancer in North-central Nigeria
Jonah Musa - 2023
Abstract
Background High risk human papillomaviruses (HR-HPV) have a causal role in cervical oncogenesis, and HIV-mediated immune suppression allows HR-HPV to persist. We studied whether vaginal microbiome community state types (CSTs) are associated with high-grade precancer and/or invasive cervical cancer (HSIL/ICC). Methods This was a cross-sectional study of adult women with cervical cancer screening (CCS) at the Jos University Teaching Hospital (JUTH) in Jos, Nigeria, between January 2020 and February 2022. Cervical swabs underwent HPV genotyping (Anyplex™ II HPV28). Cervico-vaginal lavage (CVL) sample was collected for 16 S rRNA gene amplicon sequencing. We used multivariable logistic regression modelling to assess associations between CSTs and other factors associated with HSIL/ICC. Results We enrolled 155 eligible participants, 151 with microbiome data for this analysis. Women were median age 52 (IQR:43–58), 47.7% HIV positive, and 58.1% with HSIL/ICC. Of the 138 with HPV data, 40.6% were negative for HPV, 10.1% had low-risk HPV, 26.8% had single HR-HPV, and 22.5% had multiple HR-HPV types. The overall prevalence of any HR-HPV type (single and multiple) was 49.3%, with a higher proportion in women with HSIL/ICC (NILM 31.6%, LSIL 46.5%, HSIL 40.8%, and 81.5% ICC; p = 0.007). Women with HIV were more likely to have HSIL/ICC (70.3% vs. 29.7% among women without HIV). In crude and multivariable analysis CST was not associated with cervical pathology (CST-III aOR = 1.13, CST-IV aOR = 1.31). However, in the presence of HR-HPV CST-III (aOR = 6.7) and CST-IV (aOR = 3.6) showed positive association with HSIL/ICC. Conclusion Vaginal microbiome CSTs were not significantly associated with HSIL/ICC. Our findings suggest however, that CST could be helpful in identifying women with HSIL/ICC and particularly those with HR-HPV. Characterization of CSTs using point-of-care molecular testing in women with HR-HPV should be studied as an approach to improve early detection and cervical cancer prevention. Future longitudinal research will improve our understanding of the temporal effect of non-optimal CST, HR-HPV, and other factors in cervical cancer development, prevention, and control.
Fecal Microbial Diversity of Coyotes and Wild Hogs in Texas Panhandle, USA
Babafela Awosile - 2023
Abstract
The ecology of infectious diseases involves wildlife, yet the wildlife interface is often neglected and understudied. Pathogens related to infectious diseases are often maintained within wildlife populations and can spread to livestock and humans. In this study, we explored the fecal microbiome of coyotes and wild hogs in the Texas panhandle using polymerase chain reactions and 16S sequencing methods. The fecal microbiota of coyotes was dominated by members of the phyla Bacteroidetes, Firmicutes, and Proteobacteria. At the genus taxonomic level, Odoribacter, Allobaculum, Coprobacillus, and Alloprevotella were the dominant genera of the core fecal microbiota of coyotes. While for wild hogs, the fecal microbiota was dominated by bacterial members of the phyla Bacteroidetes, Spirochaetes, Firmicutes, and Proteobacteria. Five genera, Treponema, Prevotella, Alloprevotella, Vampirovibrio, and Sphaerochaeta, constitute the most abundant genera of the core microbiota of wild hogs in this study. Functional profile of the microbiota of coyotes and wild hogs identified 13 and 17 human-related diseases that were statistically associated with the fecal microbiota, respectively (p < 0.05). Our study is a unique investigation of the microbiota using free-living wildlife in the Texas Panhandle and contributes to awareness of the role played by gastrointestinal microbiota of wild canids and hogs in infectious disease reservoir and transmission risk. This report will contribute to the lacking information on coyote and wild hog microbial communities by providing insights into their composition and ecology which may likely be different from those of captive species or domesticated animals. This study will contribute to baseline knowledge for future studies on wildlife gut microbiomes.
Foodborne Pathogens in Leafy Vegetables Grown and Consumed Locally in Yaounde, Cameroon: A Public Health Concern
Mary Nkongho Tanyitiku - 2023
Abstract
This study sought to understand the health risks of foodborne pathogens in fresh leafy vegetables that are grown and consumed locally in Yaounde, Cameroon. Through a survey, 200 respondents were recruited to relate possible food-related illnesses to leafy vegetable consumption. Additionally, a total of 168 vegetable samples consisting of six leafy vegetables and 15 irrigated water samples from five water sources were collected from farms and local markets for microbiological analysis. Using a high-fidelity DNA polymerase, five potential bacterial pathogens, namely, Shiga-toxin producing Escherichia coli (STEC), Campylobacter spp., Salmonella spp., Listeria monocytogenes and Yersinia enterocolitica were also examined. The mean counts of total viable count and total coliforms followed decreasing trends from vegetables obtained on the farms to the local markets, and these ranged from 4.98-8.74 log cfu/g and 1.77-7.42 log cfu/g respectively. All pathogens detected were of significant concern to public health showing high occurrence in some vegetables: STEC (20%) and Yersinia enterolitica (13%) in cabbage, Campylobacter spp. (21%) in lettuce, Listeria monocytogenes (15%) in African nightshade, and Salmonella spp. (15%) in amaranth. Importantly, 42% of respondents highlighted that they frequently got sick from eating leafy vegetables from the study area. These microbiological and qualitative results along with certain vegetable farming and vending practices (such as the use of untreated sewage water for crop irrigation, the sales of physically dirty, muddy, and unpackaged vegetables) indicated that foodborne diseases could be occurring among leafy vegetable-consuming populations in Cameroon.
Segmentation strategy of de novo designed four-helical bundles expands protein oligomerization modalities for cell regulation
Estera Merljak - 2023
Abstract
Protein–protein interactions govern most biological processes. New protein assemblies can be introduced through the fusion of selected proteins with di/oligomerization domains, which interact specifically with their partners but not with other cellular proteins. While four-helical bundle proteins (4HB) have typically been assembled from two segments, each comprising two helices, here we show that they can be efficiently segmented in various ways, expanding the number of combinations generated from a single 4HB. We implement a segmentation strategy of 4HB to design two-, three-, or four-chain combinations for the recruitment of multiple protein components. Different segmentations provide new insight into the role of individual helices for 4HB assembly. We evaluate 4HB segmentations for potential use in mammalian cells for the reconstitution of a protein reporter, transcriptional activation, and inducible 4HB assembly. Furthermore, the implementation of trimerization is demonstrated as a modular chimeric antigen receptor for the recognition of multiple cancer antigens.
Pilot study of a comprehensive resource estimation method from environmental DNA using universal D-loop amplification primers
Kazutoshi Yoshitake - 2023
Abstract
Many studies have investigated the ability of environmental DNA (eDNA) to identify the species. However, when individual species are to be identified, accurate estimation of their abundance using traditional eDNA analyses is still difficult. We previously developed a novel analytical method called HaCeD-Seq (haplotype count from eDNA by sequencing), which focuses on the mitochondrial D-loop sequence for eels and tuna. In this study, universal D-loop primers were designed to enable the comprehensive detection of multiple fish species by a single sequence. To sequence the full-length D-loop with high accuracy, we performed nanopore sequencing with unique molecular identifiers (UMI). In addition, to determine the D-loop reference sequence, whole genome sequencing was performed with thin coverage, and complete mitochondrial genomes were determined. We developed a UMI-based Nanopore D-loop sequencing analysis pipeline and released it as open-source software. We detected 5 out of 15 species (33%) and 10 haplotypes out of 35 individuals (29%) among the detected species. This study demonstrates the possibility of comprehensively obtaining information related to population size from eDNA. In the future, this method can be used to improve the accuracy of fish resource estimation, which is currently highly dependent on fishing catches.
Essential Oils Reduce Grey Mould Rot of Apples and Modify the Fruit Microbiome during Postharvest Storage
Giada Schiavon - 2022
Abstract
Botrytis cinerea is the causal agent of grey mould rot of apples. The efficacy of biofumigation with thyme (Thymus vulgaris), savoury (Satureja montana), and basil (Ocimum basilicum) essential oils (EOs) at 1%, 0.5%, and 0.1% concentrations were tested against B. cinerea. In vitro, the results showed 100% growth inhibition at 1% concentration for all oils. Subsequent biofumigation experiments on apples of cultivar ‘Opal’ with 1% EOs showed that, after 60 d storage, thyme and savoury EOs significantly reduced grey mould rot incidence (average incidence 2% for both treatments) compared to the control (7%). Analyses of quality indicated slightly higher fruit firmness for 1% thyme at 30 d and slightly higher titratable acidity for 1% thyme and savoury at 60 d. Sampling of the atmosphere inside the cabinets was performed to characterize and quantify the volatile components of EOs released through biofumigation. Though thymol and p-cymene were the main components of thyme EO, the antimicrobial activity was mainly due to the presence of thymol and, to a lower extent, of carvacrol. In savoury EO, carvacrol and p-cymene were the main components, whereas in basil EO, linalool and estragole were mainly present. Metabarcoding analyses showed that the epiphytic microbiome had higher richness and evenness compared to their endophytic counterpart. By the end of shelf-life, treatments with thyme EO reduced B. cinerea abundance compared to the inoculated control for both endophytes (from 36.5% to 1.5%) and epiphytes (from 7.0% to 0.7%), while favouring a significant increase in Penicillium species both in endophytes (from 0.2% to 21.5%) and epiphytes (from 0.5% to 18.6%). Results indicate that thyme EO (1%) and savoury EO (1%) are equally effective in hampering grey mould rot development in vivo.
Simple and reliable in situ CRISPR-Cas9 nuclease visualization tool is ensuring efficient editing in Streptomyces species
Alen Pšeničnik - 2022
Abstract
CRISPR-Cas9 technology has emerged as a promising tool for genetic engineering of Streptomyces strains. However, in practice, numerous technical hurdles have yet to be overcome when developing robust editing procedures. Here, we developed an extension of the CRISPR-Cas toolbox, a simple and reliable cas9 monitoring tool with transcriptional fusion of cas9 nuclease to a beta glucuronidase (gusA) visual reporter gene. The Cas9-SD-GusA tool enables in situ identification of cells expressing Cas9 nuclease following the introduction of the plasmid carrying the CRISPR-Cas9 machinery. Remarkably, when the Cas9-SD-GusA system was applied under optimal conditions, 100% of the colonies displaying GusA activity carried the target genotype. In contrast, it was shown that the cas9 sequence had undergone major recombination events in the colonies that did not exhibit GusA activity, giving rise to “escaper colonies” carrying unedited genotype. Our approach allows a simple detection of “escaper” phenotype and serves as an efficient CRISPR-Cas9 optimisation tool.
Genomic Characterisation of Canis Familiaris Papillomavirus Type 24, a Novel Papillomavirus Associated with Extensive Pigmented Plaque Formation in a Pug Dog
John S. Munday - 2022
Abstract
Numerous large dark plaques developed over the ventrum, legs and head of a 9-year-old pug dog over a 4-year-period. Histology confirmed a diagnosis of viral pigmented plaque and a short section of a novel papillomavirus (PV) type was amplified using consensus PCR primers. Taking advantage of the circular nature of PV DNA, ‘outward facing’ PCR primers allowed amplification of the full sequence. As this is the 24th PV known to infect dogs, the novel PV was designated canine papillomavirus (CPV) type 24. The CPV24 genome contained putative coding regions for 5 early proteins and 2 late ones. The CPV24 open reading frame L1 showed the highest (78.2%) similarity to CPV4 and phylogenetic analysis showed that CPV24 clustered with CPV4 and CPV16 suggesting CPV24 is the third species 2 Chipapillomavirus type identified in dogs. This is the third report of extensive pigmented plaques covering a high proportion of the skin. Both previous cases were caused CPV4 and, considering the high genetic similarity between CPV4 and CP24, infection by these CPV types may predispose to more severe clinical disease. In addition, as plaques caused by CPV16 appear more likely to progress to neoplasia, the detection of a species 2 Chipapillomavirus within a pigmented plaque may indicate the potential for more severe disease.
Essential Oils Reduce Grey Mould Rot of Apples and Modify the Fruit Microbiome during Postharvest Storage
Giada Schiavon - 2023
Abstract
Botrytis cinerea is the causal agent of grey mould rot of apples. The efficacy of biofumigation with thyme (Thymus vulgaris), savoury (Satureja montana), and basil (Ocimum basilicum) essential oils (EOs) at 1%, 0.5%, and 0.1% concentrations were tested against B. cinerea. In vitro, the results showed 100% growth inhibition at 1% concentration for all oils. Subsequent biofumigation experiments on apples of cultivar ‘Opal’ with 1% EOs showed that, after 60 d storage, thyme and savoury EOs significantly reduced grey mould rot incidence (average incidence 2% for both treatments) compared to the control (7%). Analyses of quality indicated slightly higher fruit firmness for 1% thyme at 30 d and slightly higher titratable acidity for 1% thyme and savoury at 60 d. Sampling of the atmosphere inside the cabinets was performed to characterize and quantify the volatile components of EOs released through biofumigation. Though thymol and p-cymene were the main components of thyme EO, the antimicrobial activity was mainly due to the presence of thymol and, to a lower extent, of carvacrol. In savoury EO, carvacrol and p-cymene were the main components, whereas in basil EO, linalool and estragole were mainly present. Metabarcoding analyses showed that the epiphytic microbiome had higher richness and evenness compared to their endophytic counterpart. By the end of shelf-life, treatments with thyme EO reduced B. cinerea abundance compared to the inoculated control for both endophytes (from 36.5% to 1.5%) and epiphytes (from 7.0% to 0.7%), while favouring a significant increase in Penicillium species both in endophytes (from 0.2% to 21.5%) and epiphytes (from 0.5% to 18.6%). Results indicate that thyme EO (1%) and savoury EO (1%) are equally effective in hampering grey mould rot development in vivo.
Fine-scale spatial variation shape fecal microbiome diversity and composition in black- tailed prairie dogs (Cynomys ludovicianus)
Sufia A. Neha - 2022
Abstract
Background Host associated gut microbiota are important in understanding the coevolution of host-microbe, it’s causes and consequences that may help wildlife population to adapt to its rapid climatic changes. Mammalian gut microbiota composition and diversity may be affected by a variety of factors including geographic variation, seasonal variation in diet, habitat disturbance, environmental conditions, age, and sex. However, there have been few studies that have examined how ecological and environmental factors influence gut microbiota composition in animals' natural environments. In this study, we explore how host habitat, geographical location and environmental factors affect the fecal microbiota of Cynomys ludovicianus at a small spatial scale. We collected fecal samples from five geographically distinct locations in Texas Panhandle occupying habitat classified as urban and rural areas using high throughput 16S rRNA gene amplicon sequencing. Results The results showed that microbiota of fecal samples was largely dominated by phylum Bacteroidetes. Fecal microbiome diversity and composition differed significantly across sampling sites and habitats. Prairie dogs inhabiting urban areas showed reduced fecal diversity due to more homogenous environment and anthropogenic disturbance. Urban prairie dog colonies displayed greater phylogenetic variation than those in rural habitats. Differentially abundant analysis revealed that bacterial species pathogenic to humans and animals were highly abundant in urban areas which indicates that host health and fitness might be negatively affected. Random forest model identified Alistipes shahii as the important species driving the changes in fecal microbiome composition. Despite the effects of habitat and geographic location of host, we found a strong correlation with environmental factors- average maximum temperature was the best predictor of prairie dog fecal microbial diversity. Conclusions Our findings suggest that reduction in alpha diversity in conjunction with greater dispersion in beta diversity could be indicative of declining host health in urban areas which could help determine in future conservation efforts. Moreover, several bacterial species pathogenic to humans and other animals were highly abundant in prairie dog colonies near urban areas, which may in turn adversely affect host phenotype and fitness.
A Non-Destructive High-Speed Procedure to Obtain DNA Barcodes from Soft-Bodied Insect Samples with a Focus on the Dipteran Section of Schizophora
Frederik Stein - 2022
Abstract
While the need for biodiversity research is growing, paradoxically, global taxonomical expertise is decreasing as a result of the neglected funding for young academics in taxonomy. Non-destructive approaches for DNA barcoding are necessary for a more efficient use of this dwindling expertise to fill gaps, and identify incorrect entries in sequence databases like BOLD or GenBank. They are efficient because morphological re-examination of species vouchers is still possible post-DNA barcoding. Non-destructive approaches for Diptera with a comprehensive species representation or the consideration of diagnostic fragile morphological characters are missing. Additionally, most non-destructive approaches combine a time intensive and non-destructive digestion step with common DNA extraction methods, such as commercial kits or CTAB DNA isolation. We circumvented those approaches and combined a modified non-destructive TE buffer high-speed DNA extraction, with a PCR inhibitor-resistant PCR reaction system, to a non-destructive DNA barcoding procedure for fresh and frozen samples of the Schizophora (Diptera). This method avoids morphological impairment and the application of harmful chemicals, is cost and time effective, restricts the need for laboratory equipment to a minimum, and prevents cross-contamination risk during DNA isolation. Moreover, the study indicates that the presented non-destructive DNA barcoding procedure is transferable to other soft-bodied insects. We suggest that PCR inhibitor-resistant master mixes enable the development of new—and the modification of existing—non-destructive approaches with the avoidance of further DNA template cleaning.
A diverse group of underappreciated zygnematophytes deserves in-depth exploration
Anna Busch - 2022
Abstract
The conjugating green algae (Zygnematophyceae) are the closest relatives of land plants and hence are of great evolutionary interest. Besides the popular placoderm desmids and the filamentous species, there is an underappreciated diversity of unicellular zygnematophytes with a much “simpler” morphology and smooth cell walls – traditionally referred to as “saccoderm desmids”. These saccoderm desmids have a broad geographic distribution and are ecologically diverse. Many species inhabit terrestrial habitats such as dead wood, rock surfaces and glacial ice. Furthermore, several of the saccoderm genera have turned out to be highly polyphyletic and are typically poorly captured by environmental sequencing approaches. One of these genera is Mesotaenium Nägeli, with ~70 described species and infraspecific taxa united only by a relatively simple (plate- or ribbon-like) chloroplast structure. Here, we shed some light on these inconspicuous yet important members of the algal flora and present an updated rbcL gene phylogeny of the conjugating green algae, including several new lineages of Mesotaenium-like zygnematophytes. We depict the subtle morphological differences among these lineages and discuss our updated phylogeny in the light of ecology and cell biology. In addition, we review published knowledge on photoprotective strategies of zygnematophytes, the latest insights into their evolutionary innovations, and address some technical challenges in exploring this elusive group of microalgae. Some new observations of saccoderm desmids in undersampled habitats and of their microbial associates (e.g., parasites) point to interesting avenues for future research.
Opposite effects of stress on effortful motivation in high and low anxiety are mediated by CRHR1 in the VTA
Ioannis Zalachoras - 2022
Abstract
Individuals frequently differ in their behavioral and cognitive responses to stress. However, whether motivation is differently affected by acute stress in different individuals remains to be established. By exploiting natural variation in trait anxiety in outbred Wistar rats, we show that acute stress facilitates effort-related motivation in low anxious animals, while dampening effort in high anxious ones. This model allowed us to address the mechanisms underlying acute stress–induced differences in motivated behavior. We show that CRHR1 expression levels in dopamine neurons of the ventral tegmental area (VTA)—a neuronal type implicated in the regulation of motivation—depend on animals’ anxiety, and these differences in CRHR1 expression levels explain the divergent effects of stress on both effortful behavior and the functioning of mesolimbic DA neurons. These findings highlight CRHR1 in VTA DA neurons—whose levels vary with individuals’ anxiety—as a switching mechanism determining whether acute stress facilitates or dampens motivation.
Bacillus velezensis Identification and Recombinant Expression, Purification, and Characterization of Its Alpha-Amylase
Xiaodong Zhang - 2021
Abstract
Amylases account for about 30% of the global market of industrial enzymes, and the current amylases cannot fully meet industrial needs. This study aimed to identify a high α-amylase producing bacterium WangLB, to clone its α-amylase coding gene, and to characterize the α-amylase. Results showed that WangLB belonged to Bacillus velezensis whose α-amylase gene was 1980 bp coding 659 amino acids designated as BvAmylase. BvAmylase was a hydrophilic stable protein with a signal peptide and a theoretical pI of 5.49. The relative molecular weight of BvAmylase was 72.35 kDa, and was verified by SDS-PAGE. Its modeled structure displayed that it was a monomer composed of three domains. Its optimum temperature and pH were 70 °C and pH 6.0, respectively. It also showed high activity in a wide range of temperatures (40–75 °C) and a relatively narrow pH (5.0–7.0). It was a Ca2+-independent enzyme, whose α-amylase activity was increased by Co2+, Tween 20, and Triton X-100, and severely decreased by SDS. The Km and the Vmax of BvAmylase were 3.43 ± 0.53 and 434.19 ± 28.57 U/mg. In conclusion, the α-amylase producing bacterium WangLB was identified, and one of its α-amylases was characterized, which will be a candidate enzyme for industrial applications.
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Customer Testimonials

repliQa HiFi ToughMix
"Worked well with our 16s amplicon set up. Clean products, no extraneous peaks."
Next Generation Genomics Specialist | Virginia Tech
repliQa HiFi ToughMix

"Generated 625 bp fragment w/o errors (sequence-confirmed) using 3-step protocol. Worked ~3 times faster than conventional method"

Professor | Vanderbilt University
repliQa HiFi ToughMix

" After struggling to amplify a roughly 5 kb fragment, I tested the repliQa HiFi ToughMix on the same template using a two step PCR per the suggested protocol. It amplified on the first try. Perhaps the most impressive part was the speed. With the suggested extension time of 5 seconds per kb, total reaction time was reduced by two hours compared to the previous polymerase. Subsequent cloning and sequencing showed the amplified fragment contained zero errors. I was quite pleased with this product. "

Gary G. | Scientist, OMRF
repliQa HiFi ToughMix

"All I can say from testing repliQa HiFi ToughMix is that the results are jaw-dropping. The Platinum™ SuperFi II PCR Master Mix did nothing in the same amount of time under the same conditions. The assay I was testing out is a new way to amplify entire mitogenomes as a single amplicon (~15kb). This is a game changer, cutting the PCR time from ~8.5hrs to 1.5hrs, and way more product is generated. I am not often floored by data, but these results are amazing. I have another project I am working on with this assay and I am going to switch to this polymerase for sure. Good work Quantabio!"

A. K. | Post Doc, BCM
repliQa HiFi ToughMix

"The kit have a great power of amplification and extreme fast speed, I just need one hour to get result which I usually spent three hours before."

Simon S. | Professor, Tennessee State University
repliQa HiFi ToughMix

"This can amplify the tough template for SURE. I was struggled with the tough template that showed poor quality results with other many PCR enzymes. This repliQa saved my life and eventually gave me the clear and strong band by gel."

Lili C. | Post Doc, USC
repliQa HiFi ToughMix

"I used NEB Q5 for the same plasmid for PCR, same annealing temperature. Only the repliQa HiFi toughMix gave me the correct size products. It is very fast and works much better than NEB Q5 for difficult plasmids in PCR."

Dr. Kara C. | UT Southwestern Medical Center
repliQa HiFi ToughMix

"Worked great amplifying two gene target from plasmid DNA. It was the first time I have performed a two step reaction and it worked great. It was awesome running everything in 23 minutes too."

Dr. Brandon L. | University of Nebraska

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