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BioTechniques

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https://www.readbyqxmd.com/read/30014738/a-bioinformatics-approach-to-identify-telomere-sequences
#1
Indira Somanathan, Chris Baysdorfer
Conventional approaches to identify a telomere motif in a new genome are laborious and time-intensive. An efficient new methodology based on next-generation sequencing (NGS), de novo sequence repeat finder (SERF) and fluorescence in situ hybridization (FISH) is presented. Unlike existing heuristic approaches, SERF utilizes an exhaustive analysis of raw NGS reads or assembled contigs for rapid de novo detection of conserved tandem repeats representing telomere motifs. SERF was validated using the NGS data from Ipheion uniflorum and Allium cepa with known telomere motifs...
July 2018: BioTechniques
https://www.readbyqxmd.com/read/30014737/long-term-imaging-of-calcium-dynamics-using-genetically-encoded-calcium-indicators-and-automatic-tracking-of-cultured-cells
#2
Igor Razlivanov, Teresa Liew, Eira Watts Moore, Alaa Al-Kathiri, Tayma Bartram, Dmitriy Kuvshinov, Anton Nikolaev
Calcium dynamics is crucial for many signaling pathways and cell functions. Understanding how calcium regulates cell function often requires long-term imaging of calcium dynamics. Here we report a methodological approach of long-term (5-10 h) imaging of calcium dynamics in cultured cells. The approach links calcium imaging using genetically encoded calcium indicators and semi-automatic tracking of individual cells. It can be used in a large variety of situations, ranging from the role of calcium in biological processes to cell heterogeneity and screening of drugs modifying signaling pathways...
July 2018: BioTechniques
https://www.readbyqxmd.com/read/30014736/non-lethal-method-for-the-preparation-of-metaphase-spreads-using-cultured-mantle-tissue-from-live-adult-abalone
#3
Jun Hyung Ryu, Sang Yoon Lee, Yoon Kwon Nam, Seung Pyo Gong
Metaphase spread preparation in adult abalone has not been successful, which has restricted the applications of karyotyping-based technologies. Here, we present a non-lethal method to enable preparation of metaphase spreads from live adult abalone using a tissue culture method. Mantle tissue fragments from live adult abalone were cultured in vitro and the cultured cells were used for metaphase spread preparation. To retrieve a sufficient number of proliferating cells required for metaphase spread preparation, at least 14 days of culture was required, and culturing the marginal zone of mantle was more optimal than culturing other areas...
July 2018: BioTechniques
https://www.readbyqxmd.com/read/30014735/designer-microbiomes
#4
Alfie Gleeson, Abigail Sawyer
To what extent can your microbiome define you and how can it be altered to optimize your health?
July 2018: BioTechniques
https://www.readbyqxmd.com/read/30014734/luciferase-complementation-based-detection-of-g-protein-coupled-receptor-activity
#5
Hideaki Yano, Ning Sheng Cai, Jonathan A Javitch, Sergi Ferré
Protein complementation assays (PCA) are used as pharmacological tools, enabling a wide array of applications, ranging from studies of protein-protein interactions to second messenger effects. Methods to detect activities of G protein-coupled receptors (GPCRs) have particular relevance for drug screening. Recent development of an engineered luciferase NanoLuc created the possibility of generating a novel PCA, which in turn could open a new avenue for developing drug screening assays. Here we identified a novel split position for NanoLuc and demonstrated its use in a series of fusion constructs to detect the activity of GPCRs...
July 2018: BioTechniques
https://www.readbyqxmd.com/read/30014733/microgels-and-apparatus-for-page-of-nucleic-acids-in-one-or-two-dimensions
#6
Hans G Thormar, Gudmundur H Gunnarsson, Bjarki Gudmundsson, Kristjan Leosson, Peter Estibeiro, Jon J Jonsson
We describe a system for horizontal 1D or 2D PAGE comprising an apparatus and microgels. There is no buffer outside the gel, making handling and sample loading easy. Specially designed electrodes on all four sides allow 2D electrophoresis without gel rotation. Electrophoresis is completed within 20 min and sensitivity is in the subnanogram range. The system is temperature controlled for speed, denaturation of nucleic acid molecules and maintaining molecules single-stranded. The system allows characterization of structure, conformation and damage in complex nucleic acid preparations...
July 2018: BioTechniques
https://www.readbyqxmd.com/read/30014732/could-closing-the-gender-gap-in-stemm-really-take-a-quarter-of-a-millennium
#7
Francesca Lake
No abstract text is available yet for this article.
July 2018: BioTechniques
https://www.readbyqxmd.com/read/30014731/inorganic-lanthanides-induce-pcr-bias-towards-shorter-amplicons
#8
Fan Li, Hongwei Sun, Shuke Yang, Rui Gao, Xiao Hui Xu, Xingbo Lu
Rare earth elements have many uses, and are frequently included in products such as fluorescent materials, hydride batteries, catalytic materials and lasers. In this study, it was observed that trivalent lanthanide ions (Ln[III] ions) appeared to inhibit the synthesis of large fragments in PCR assays, thus resulting in the preferential amplification of shorter sequences. It is therefore speculated that this Ln(III) ion-mediated bias could be utilized to improve the success rates for amplification of shorter products...
July 2018: BioTechniques
https://www.readbyqxmd.com/read/30014730/generation-of-flp-in-tm-ready-dg44-and-lec-3-2-8-1-cho-cell-lines-for-quick-and-easy-constitutive-protein-expression
#9
D C Soler, A E Young, A Vahedi-Faridi, T S McCormick
The well-characterized cell line Chinese hamster ovary (CHO) has been used to produce numerous biopharmaceuticals and is an important tool for basic research. However, introducing foreign DNA into specially modified CHO cells such as DG44 and Lec 3.2.8.1 can sometimes be an arduous process. Here we show that the Flp-intm plasmid can be modified to produce a fluorescent tracer protein tag (mCherrytm ) as a fusion reporter, to allow for the rapid selection of single-cell sorted, isogenic Flp-intm -ready DG44 and Lec 3...
July 2018: BioTechniques
https://www.readbyqxmd.com/read/29939094/determination-of-polyurethane-grafted-peptide-gsgredvgsg-using-bicinchoninic-acid-assay
#10
Beata A Butruk-Raszeja, Aleksandra Kuźmińska
The goal in the presented study was to develop a simple, fast and accurate method for measuring the surface density of a short peptide sequence bound to a polymeric substrate. We analyzed polyurethane samples chemically modified with acrylic acid and polyurethane-grafted peptide (GSGREDVGSG) and investigated the possibility of using the bicinchoninic acid (BCA) assay to determine surface density of the solid-supported peptide. We set the conditions (temperature, time) under which the test should be conducted...
June 2018: BioTechniques
https://www.readbyqxmd.com/read/29939093/the-flash-small-pool-pcr-how-to-transform-blotting-and-numerous-hybridization-steps-into-a-simple-denatured-pcr
#11
Elodie Dandelot, Geneviève Gourdon
Numerous human diseases are associated with abnormal expansion of unstable trinucleotide repeats (TNRs). TNR instability mechanisms are complex, and remain only partially understood. Small-pool-PCR (SP-PCR) is the reference method to assess TNR instability. SP-PCR amplifies a low number of DNA molecules and is followed by Southern blot. However, SP-PCR remains expensive and time consuming. Here, we describe an optimized SP-PCR that can be done in a day, which reduces cost and experimental biases: the flash-small-pool PCR (FSP-PCR)...
June 2018: BioTechniques
https://www.readbyqxmd.com/read/29939092/thanking-biotechniques-advertisers-for-making-print-possible
#12
Francesca Lake
No abstract text is available yet for this article.
June 2018: BioTechniques
https://www.readbyqxmd.com/read/29939091/a-stainless-steel-mortar-pestle-and-sleeve-design-for-the-efficient-fragmentation-of-ancient-bone
#13
Agata T Gondek, Sanne Boessenkool, Bastiaan Star
Different types of milling equipment - such as oscillating ball mills, freezer mills, mortar and pestle - can be used to fragment ancient bone prior to DNA extraction. However, each of these tools is associated with practical drawbacks. Here, we present the design for a stainless-steel mortar and pestle, with a removable sleeve to contain bone material. The tool is easy to clean, practical and its simplicity allows university workshops equipped with a lathe, boring tools and a milling machine to make these components at local expense...
June 2018: BioTechniques
https://www.readbyqxmd.com/read/29939090/a-simple-microfluidic-device-for-live-cell-imaging-of-arabidopsis-cotyledons-leaves-and-seedlings
#14
Shia Vang, Kati Seitz, Patrick J Krysan
One of the challenges of performing live-cell imaging in plants is establishing a system for securing the sample during imaging that allows for the rapid addition of treatments. Here we report how a commercially available device called a HybriWell™ can be repurposed to create an imaging chamber suitable for Arabidopsis seedlings, cotyledons and leaves. Liquid in the imaging chamber can be rapidly exchanged to introduce chemical treatments via microfluidic passive pumping. When used in conjunction with fluorescent biosensors, this system can facilitate live-cell imaging studies of signal transduction pathways triggered by different treatments...
June 2018: BioTechniques
https://www.readbyqxmd.com/read/29939089/crispr-cas9-the-gold-standard-of-genome-editing
#15
Alfie Gleeson, Abigail Sawyer
No abstract text is available yet for this article.
June 2018: BioTechniques
https://www.readbyqxmd.com/read/29939088/a-page-screening-approach-for-identifying-crispr-cas9-induced-mutations-in-zebrafish
#16
Ariel J VanLeuven, Sungdae Park, Douglas B Menke, James D Lauderdale
The introduction of CRISPR-Cas9 technology for targeted mutagenesis has revolutionized reverse genetics and made genome editing a realistic option in many model organisms. One of the difficulties with this technique is screening for mutations in large numbers of samples. Many screening approaches for identifying CRISPR-Cas9 mutants have been published; however, in practice these methods are time consuming, expensive, or often yield false positives. This report describes a PCR-based screening approach using non-denaturing PAGE...
June 2018: BioTechniques
https://www.readbyqxmd.com/read/29939087/absolute-bioluminescence-imaging-at-the-single-cell-level-with-a-light-signal-at-the-attowatt-level
#17
Toshiteru Enomoto, Hidehiro Kubota, Kaneo Mori, Masahiro Shimogawara, Masahiro Yoshita, Yoshihiro Ohmiya, Hidefumi Akiyama
Bioluminescence imaging (BLI) demonstrates cellular events as a light signal at the single-cell level using a highly sensitive, cooled CCD camera. However, BLI signals are relative values and thus, images taken on different days or using different equipment cannot be compared directly. We established a reference LED light source that was characteristic of the total flux and light distribution and calibrated the BLI system as an absolute light signal. This calibrated BLI system revealed that the average light signal of beetle luciferase was at an attowatt level per sec at the single cell level...
June 2018: BioTechniques
https://www.readbyqxmd.com/read/29793364/you-asked-we-listened-get-ready-for-more-tips-tricks-discussion-from-biotechniques
#18
Francesca Lake
No abstract text is available yet for this article.
May 2018: BioTechniques
https://www.readbyqxmd.com/read/29793363/detection-of-proteolytic-activity-by-covalent-tethering-of-fluorogenic-substrates-in-zymogram-gels
#19
Ameya A Deshmukh, Jessica L Weist, Jennifer L Leight
Current zymographic techniques detect only a subset of known proteases due to the limited number of native proteins that have been optimized for incorporation into polyacrylamide gels. To address this limitation, we have developed a technique to covalently incorporate fluorescently labeled, protease-sensitive peptides using an azido-PEG3-maleimide crosslinker. Peptides incorporated into gels enabled measurement of MMP-2, -9, -14, and bacterial collagenase. Sensitivity analysis demonstrated that use of peptide functionalized gels could surpass detection limits of current techniques...
May 2018: BioTechniques
https://www.readbyqxmd.com/read/29793362/quantifying-cell-free-dna-in-urine-comparison-between-commercial-kits-impact-of-gender-and-inter-individual-variation
#20
Greta Streleckiene, Hayley M Reid, Norbert Arnold, Dirk Bauerschlag, Michael Forster
DNA can enter the blood circulation from living cells by extracellular vesicles or at cell death, and pass into urine through the kidney barrier. Urine can be collected non-invasively, making it an interesting source of cell-free DNA (cfDNA) for research studies and ultimately for clinical diagnostics. However, there is currently a lack of data on the quantity and variability of cfDNA in urine. Here, we benchmark two commercial urine cfDNA isolation kits with respect to the quantity of DNA, the labor time, and cost...
May 2018: BioTechniques
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