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BioTechniques

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https://www.readbyqxmd.com/read/27938324/analysis-and-quantification-of-in-vitro-myoblast-fusion-using-the-ladd-multiple-stain
#1
Rhys McColl, Mthokozisi Nkosi, Celia Snyman, Carola Niesler
Myoblast fusion, which is essential for muscle development, regeneration, and repair, can be assessed in vitro via the calculation of a fusion index. Traditionally, this requires use of either immunocytochemistry or fluorescently-labeled cytoskeletal staining, followed by microscopy and laborious analysis. The expense and time-consuming nature of the optimization and application of antibody-based techniques such as immunocytochemistry, as well as the need for specialized analytical equipment such as fluorescence microscopes, presents a barrier to the routine analysis of this crucial step during terminal differentiation...
December 1, 2016: BioTechniques
https://www.readbyqxmd.com/read/27938323/targeted-capture-and-sequencing-of-gene-sized-dna-molecules
#2
Michael Giolai, Pirita Paajanen, Walter Verweij, Lawrence Percival-Alwyn, David Baker, Kamil Witek, Florian Jupe, Glenn Bryan, Ingo Hein, Jonathan D G Jones, Matthew D Clark
Targeted capture provides an efficient and sensitive means for sequencing specific genomic regions in a high-throughput manner. To date, this method has mostly been used to capture exons from the genome (the exome) using short insert libraries and short-read sequencing technology, enabling the identification of genetic variants or new members of large gene families. Sequencing larger molecules results in the capture of whole genes, including intronic and intergenic sequences that are typically more polymorphic and allow the resolution of the gene structure of homologous genes, which are often clustered together on the chromosome...
December 1, 2016: BioTechniques
https://www.readbyqxmd.com/read/27938322/protein-consensus-based-surface-engineering-procos-a-computer-assisted-method-for-directed-protein-evolution
#3
Amol V Shivange, Hans Wolfgang Hoeffken, Stefan Haefner, Ulrich Schwaneberg
Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: (i) identification of conserved and highly variable regions; (ii) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; (iii) in vitro DNA recombination of synthetic genes; and (iv) screening for active variants...
December 1, 2016: BioTechniques
https://www.readbyqxmd.com/read/27938321/high-yield-recombinant-expression-and-purification-of-marginally-soluble-short-elastin-like-polypeptides
#4
Markian S Bahniuk, Abdullah K Alshememry, Larry D Unsworth
The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC)...
December 1, 2016: BioTechniques
https://www.readbyqxmd.com/read/27938320/-letter-to-the-editor-many-commercial-hot-start-polymerases-demonstrate-activity-prior-to-thermal-activation
#5
Aaron J Stevens, Sarah Appleby, Martin A Kennedy
Address correspondence to Martin A. Kennedy, Department of Pathology & Carney Centre for Pharmacogenomics, University of Otago, Christchurch, PO Box 4345, Christchurch, New Zealand. E-mail: martin.kennedy@otago.ac.nz.
December 1, 2016: BioTechniques
https://www.readbyqxmd.com/read/27938319/larger-sharper-deeper
#6
Sarah Webb
Microscopy continues to expand our knowledge of brain structure and function. Sarah Webb looks at some of the latest tools and techniques leading the charge.
December 1, 2016: BioTechniques
https://www.readbyqxmd.com/read/27712584/a-cost-effective-method-to-immobilize-hydrated-soft-tissue-samples-for-atomic-force-microscopy
#7
Suchit Sahai, Marysuna Wilkerson, Ana Maria Zaske, Scott D Olson, Charles S Cox, Fabio Triolo
Immobilizing hydrated soft tissue specimens for atomic force microscopy (AFM) is a challenge. Here, we describe a simple and very cost-effective immobilization method, based on the use of transglutaminase in an aqueous environment, and successfully apply it to AFM characterization of human native Wharton's Jelly (nWJ), the gelatinous connective tissue matrix of the umbilical cord. A side-by-side comparison with a widely used polyphenolic protein-based tissue adhesive (Corning Cell-Tak), which is known to bind strongly to virtually all inorganic and organic surfaces in aqueous environments, shows that both adhesives successfully immobilize nWJ in its physological hydrated state...
October 1, 2016: BioTechniques
https://www.readbyqxmd.com/read/27712583/extraction-of-high-molecular-weight-genomic-dna-for-long-read-sequencing-of-single-molecules
#8
Baptiste Mayjonade, Jérôme Gouzy, Cécile Donnadieu, Nicolas Pouilly, William Marande, Caroline Callot, Nicolas Langlade, Stéphane Muños
De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose...
October 1, 2016: BioTechniques
https://www.readbyqxmd.com/read/27712582/spotsizer-high-throughput-quantitative-analysis-of-microbial-growth
#9
Leanne Bischof, Martin Převorovský, Charalampos Rallis, Daniel C Jeffares, Yulia Arzhaeva, Jürg Bähler
Microbial colony growth can serve as a useful readout in assays for studying complex genetic interactions or the effects of chemical compounds. Although computational tools for acquiring quantitative measurements of microbial colonies have been developed, their utility can be compromised by inflexible input image requirements, non-trivial installation procedures, or complicated operation. Here, we present the Spotsizer software tool for automated colony size measurements in images of robotically arrayed microbial colonies...
October 1, 2016: BioTechniques
https://www.readbyqxmd.com/read/27712581/optimization-of-diamond-nucleic-acid-dye-for-quantitative-pcr
#10
Alicia M Haines, Shanan S Tobe, Adrian Linacre
Here, we evaluate Diamond Nucleic Acid Dye (DD) for use in quantitative PCR (qPCR) applications. Although DD is a commercially available stain for detection of DNA separated by gel electrophoresis, its use as a detection dye in qPCR has yet to be described. To determine if DD can be used in qPCR, we investigated its inhibitory effects on qPCR at concentrations ranging 0.1-2.5×. Serial dilution of DNA was used to determine the efficiency, sensitivity, and linearity of DD-generated qPCR data in comparison to other commonly used fluorescent dyes such as SYBR Green (SG), EvaGreen (EG), and BRYT Green (BG)...
October 1, 2016: BioTechniques
https://www.readbyqxmd.com/read/27712580/pcr-amplification-of-gc-rich-dna-regions-using-the-nucleotide-analog-n4-methyl-2-deoxycytidine-5-triphosphate
#11
Cyntia R Flores-Juárez, Eva González-Jasso, Anaid Antaramian, Reynaldo C Pless
GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons...
October 1, 2016: BioTechniques
https://www.readbyqxmd.com/read/27071609/step-by-step-protocol-to-perfuse-and-dissect-the-mouse-parotid-gland-and-isolation-of-high-quality-rna-from-murine-and-human-parotid-tissue
#12
Christoph Watermann, Klaus Peter Valerius, Steffen Wagner, Claus Wittekindt, Jens Peter Klussmann, Eveline Baumgart-Vogt, Srikanth Karnati
Macroscopic identification and surgical removal of the mouse parotid gland is demanding because of its anatomic location and size. Moreover, the mouse parotid gland contains high concentrations of RNases, making it difficult to isolate high-quality RNA. So far, appropriate methods for optimal perfusion-fixation and dissection of mouse parotid glands, as well as the isolation of high quality RNA from this tissue, are not available. Here we present a simple, optimized, step-by-step surgical method to perfuse and isolate murine parotid glands...
April 2016: BioTechniques
https://www.readbyqxmd.com/read/27071608/bioanalyzer-chips-can-be-used-interchangeably-for-many-analyses-of-dna-or-rna
#13
Jessica Davies, Tom Denyer, James Hadfield
The Agilent 2100 Bioanalyzer enables small-scale gel electrophoretic separation of nucleic acids on a microfluidic chip; however, a shortage of chips and an excess of reagents are common issues. Here we explore the compatibility of two commonly used Bioanalyzer reagents with three Bioanalyzer chip types. Microfluidic electrophoretic separation of DNA and RNA using DNA High Sensitivity and RNA 6000 Nano reagents, respectively, was successfully performed on multiple chip types following the assay-specific protocols...
April 2016: BioTechniques
https://www.readbyqxmd.com/read/27071607/aminoxytmt-a-novel-multi-functional-reagent-for-characterization-of-protein-carbonylation
#14
Somaieh Afiuni-Zadeh, John C Rogers, Sergei I Snovida, Ryan D Bomgarden, Timothy J Griffin
Protein carbonylation is a common oxidative stress (OS)-driven post-translational modification (PTM). Proteome-wide carbonylation events can best be characterized using a combination of analytical approaches. Immunoblotting of carbonylated proteins provides data on the extent of modifications within complex samples, as well as a broad comparison of carbonylation profiles between different biological states (e.g., disease versus control), while mass spectrometry (MS)-based analysis provides information on proteins susceptible to carbonylation, as well as the potential for quantitative characterization of specific sites of amino acid modification...
April 2016: BioTechniques
https://www.readbyqxmd.com/read/27071606/mutant-dna-quantification-by-digital-pcr-can-be-confounded-by-heating-during-dna-fragmentation
#15
Qing Kang, Brian Parkin, Maria D Giraldez, Muneesh Tewari
Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance. Wild-type (WT) human genomic DNA was fragmented by heating, restriction digestion, or acoustic shearing using a Covaris focused-ultrasonicator...
April 2016: BioTechniques
https://www.readbyqxmd.com/read/27071605/targeted-reduction-of-highly-abundant-transcripts-using-pseudo-random-primers
#16
Ophélie Arnaud, Sachi Kato, Stéphane Poulain, Charles Plessy
Transcriptome studies based on quantitative sequencing can estimate levels of gene expression by measuring target RNA abundance in sequencing libraries. Sequencing costs are proportional to the total number of sequenced reads, and in order to cover rare RNAs, considerable quantities of abundant and identical reads are needed. This major limitation can be addressed by depleting a proportion of the most abundant sequences from the library. However, such depletion strategies involve either extra handling of the input RNA sample or use of a large number of reverse transcription primers, termed not-so-random (NSR) primers, which are costly to synthesize...
April 2016: BioTechniques
https://www.readbyqxmd.com/read/27071604/democratizing-mass-spectrometry
#17
Jeffrey Perkel
Mass spectrometry is often seen as a complicated, highly technical technique requiring years of experience to master. Jeffrey Perkel talks to users about the best ways for novices to approach mass spectrometry experiments.
April 2016: BioTechniques
https://www.readbyqxmd.com/read/26956087/making-sense-of-big-data
#18
Jeffrey Perkel
As data sets grow in size, new computational tools are needed. Jeffrey Perkel looks at the ins and outs of data analysis.
March 2016: BioTechniques
https://www.readbyqxmd.com/read/27257654/honoring-technology-developers
#19
EDITORIAL
(no author information available yet)
No abstract text is available yet for this article.
February 2016: BioTechniques
https://www.readbyqxmd.com/read/26842356/direct-current-electrical-stimulation-chamber-for-treating-cells-in-vitro
#20
Sahba Mobini, Liudmila Leppik, John H Barker
Electrical stimulation has been shown to promote healing and regeneration in skin, bone, muscle, and nerve tissues in clinical studies. Recently, studies applying electrical stimulation to influence cell behavior associated with proliferation, differentiation, and migration have provided a better understanding of the underlying mechanisms of electrical stimulation-based clinical treatments and improved tissue-engineered products through electro-bioreactor technologies. Here, we present a novel device for delivering direct current (DC) electrical stimulation (ES) to cultivated cells in vitro...
February 2016: BioTechniques
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