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BioTechniques

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https://www.readbyqxmd.com/read/28298179/preparation-of-peptide-mhc-and-t-cell-receptor-dextramers-by-biotinylated-dextran-doping
#1
Michael T Bethune, Begoña Comin-Anduix, Yu-Hsien Hwang Fu, Antoni Ribas, David Baltimore
Peptide-major histocompatibility complex (pMHC) multimers enable the detection, characterization, and isolation of antigen-specific T-cell subsets at the single-cell level via flow cytometry and fluorescence microscopy. These labeling reagents exploit a multivalent scaffold to increase the avidity of individually weak T-cell receptor (TCR)-pMHC interactions. Dextramers are an improvement over the original streptavidin-based tetramer technology because they are more multivalent, improving sensitivity for rare, low-avidity T cells, including self/tumor-reactive clones...
March 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28298178/antibody-incubation-at-37%C3%A2-c-improves-fluorescent-immunolabeling-in-free-floating-thick-tissue-sections
#2
Xia Xiao, Ya-Ping Feng, Bin Du, Han-Ru Sun, You-Quan Ding, Jian-Guo Qi
Fluorescent immunolabeling and imaging in free-floating thick (50-60 μm) tissue sections is relatively simple in practice and enables design-based non-biased stereology, or 3-D reconstruction and analysis. This method is widely used for 3-D in situ quantitative biology in many areas of biological research. However, the labeling quality and efficiency of standard protocols for fluorescent immunolabeling of these tissue sections are not always satisfactory. Here, we systematically evaluate the effects of raising the conventional antibody incubation temperatures (4°C or 21°C) to mammalian body temperature (37°C) in these protocols...
March 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28298177/tympanic-membrane-organ-culture-using-cell-culture-well-inserts-engrafted-with-tympanic-membrane-tissue-explants
#3
Lawrence J Liew, Richard M Day, Rodney J Dilley
Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks...
March 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28298176/semi-automated-tip-snip-cloning-of-restriction-fragments-into-and-out-of-plasmid-polylinkers
#4
Ichiro Matsumura
Synthetic biologists rely on semi-synthetic recombinant plasmids, but DNA synthesis is constrained by practical limits on length, accuracy, and sequence composition. Cloned DNA parts can be assembled into longer constructs via subcloning, but conventional methods are labor-intensive. One-pot recombination reactions are more convenient but harder to troubleshoot, and those that depend on PCR to create fragments with compatible ends necessitate re-sequencing. The Tip Snip protocol described here enables the subcloning of an insert from one plasmid polylinker into another without PCR or gel purification steps...
March 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28298175/cells-in-the-third-dimension
#5
Sarah Webb
Sarah Webb explores the expanding tools and technologies for 3-D cell culture-from hydrogels to organs-on-a-chip.
March 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193152/a-fast-method-for-analyzing-essential-protein-mutants-in-human-cells
#6
Frank Dietsch, Mariel Donzeau, Agnes M Cordonnier, Etienne Weiss, Bruno Chatton, Marc Vigneron
Here we developed a complementation method for the study of essential genes in live human cells using the CRISPR/Cas9 system. Proteins encoded by essential genes were expressed using a derivative of the pCEP4 compensating plasmid in combination with Cas9 endonuclease targeting of the chromosomal genes. We show that this strategy can be applied to essential genes, such as those coding for proliferating cell nuclear antigen (PCNA) and DNA polymerase delta subunit 2 (POLD2). As demonstrated for the PCNA protein, our method allows mutational analysis of essential protein-coding sequences in live cells...
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193151/extraction-of-ultrashort-dna-molecules-from-herbarium-specimens
#7
Rafal M Gutaker, Ella Reiter, Anja Furtwängler, Verena J Schuenemann, Hernán A Burbano
DNA extracted from herbarium specimens is highly fragmented; therefore, it is crucial to use extraction protocols that retrieve short DNA molecules. Improvements in extraction and DNA library preparation protocols for animal remains have allowed efficient retrieval of molecules shorter than 50 bp. Here, we applied these improvements to DNA extraction protocols for herbarium specimens and evaluated extraction performance by shotgun sequencing, which allows an accurate estimation of the distribution of DNA fragment lengths...
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193150/maleimide-scavenging-enhances-determination-of-protein-s-palmitoylation-state-in-acyl-exchange-methods
#8
Charlotte H Hurst, Dionne Turnbull, Fiona Plain, William Fuller, Piers A Hemsley
S-palmitoylation (S-acylation) is emerging as an important dynamic post-translational modification of cysteine residues within proteins. Current assays for protein S-palmitoylation involve either in vivo labeling or chemical cleavage of S-palmitoyl groups to reveal a free cysteine sulfhydryl that can be subsequently labeled with an affinity handle (acyl-exchange). Assays for protein S-palmitoylation using acyl-exchange chemistry therefore require blocking of non-S-palmitoylated cysteines, typically using N-ethylmaleimide (NEM), to prevent non-specific detection...
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193149/taqman-probes-as-blocking-agents-for-enriched-pcr-amplification-and-dna-melting-analysis-of-mutant-genes
#9
Irina V Botezatu, Irina O Panchuk, Anna M Stroganova, Anastasia I Senderovich, Valentina N Kondratova, Valery P Shelepov, Anatoly V Lichtenstein
Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ~5% and 15%-20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR...
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193148/external-calibration-with-drosophila-whole-cell-spike-ins-delivers-absolute-mrna-fold-changes-from-human-rna-seq-and-qpcr-data
#10
Franziska Taruttis, Maren Feist, Phillip Schwarzfischer, Wolfram Gronwald, Dieter Kube, Rainer Spang, Julia C Engelmann
Gene expression measurements are typically performed on a fixed-weight aliquot of RNA, which assumes that the total number of transcripts per cell stays nearly constant across all conditions. In cases where this assumption does not hold (e.g., when comparing cell types with different cell sizes) the expression data provide a distorted view of cellular events. Assuming constant numbers of total transcripts, increases in expression of some RNAs must be compensated for by decreases in expression of others. Therefore, we propose calibrating gene expression data to an external reference point, the number of cells in the sample, using whole-cell spike-ins...
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28193147/print-on-demand
#11
Sarah Webb
Advances in 3-D printing and biomaterials are helping to shape the field of regenerative medicine. Sarah Webb examines the latest developments.
February 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28118814/bxb1-phage-recombinase-assists-genome-engineering-in-drosophila-melanogaster
#12
Roumen Voutev, Richard S Mann
Rapid and reliable genome modifications provide the basis for detailed in vivo functional analysis of any genomic entity (gene, regulatory DNA, non-coding RNA, etc). With the advent of CRISPR/Cas9 genome editing technology, manipulation of a particular genomic locus has become a routine undertaking in variety of model organisms, including the fruit fly Drosophila melanogaster. To further diversify the available tools for genome engineering, we successfully harnessed the phage recombinase Bxb1 to perform recombinase-mediated cassette exchange (RMCE) in D...
January 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28118813/tumblescore-run-and-tumble-analysis-for-low-frame-rate-motility-videos
#13
Alex Eli Pottash, Ryan McKay, Chelsea R Virgile, Hana Ueda, William E Bentley
Scientists often exploit the motility of peritrichously flagellated bacteria for various applications. A common alteration is modifying the frequency of mid-movement changes in direction, known as tumbles. Such differences in bacterial swimming patterns can prove difficult to quantify, especially for those without access to high-speed optical equipment. Traditionally, scientists have resorted to less accurate techniques, such as soft agar plate assays, or have been forced to invest in costly equipment. Here, we present TumbleScore, software designed to track and quantify bacterial movies with slow, as well as fast, frame-rates...
January 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28118812/a-practical-guide-to-filtering-and-prioritizing-genetic-variants
#14
Mahjoubeh Jalali Sefid Dashti, Junaid Gamieldien
Next-generation sequencing (NGS) of whole genomes and exomes is a powerful tool in biomedical research and clinical diagnostics. However, the vast amount of data produced by NGS introduces new challenges and opportunities, many of which require novel computational and theoretical approaches when it comes to identifying the causal variant(s) for a disease of interest. While workflows and associated software to process raw data and produce high-confidence variant calls have significantly improved, filtering tens of thousands of candidates to identify a subset relevant to a specific study is still a complex exercise best left to bioinformaticists...
January 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28118811/-letter-to-the-editor-isolation-of-mitochondria-is-necessary-for-precise-quantification-of-mitochondrial-dna-damage-in-human-carcinoma-samples
#15
Arianna Barchiesi, Umberto Baccarani, Blase Billack, Gianluca Tell, Carlo Vascotto
Address correspondence to Carlo Vascotto, Department of Medical and Biological Sciences, University of Udine, Udine, 33100, Italy. E-mail: carlo.vascotto@uniud.it.
January 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28118810/big-labs-and-tiny-instruments
#16
Nathan S Blow
At many research institutions, lab space is more valuable than gold. Developers are taking note by designing smaller instruments with enhanced capabilities. Nathan Blow looks inside today's tiny lab.
January 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/27938324/analysis-and-quantification-of-in-vitro-myoblast-fusion-using-the-ladd-multiple-stain
#17
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
#18
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
#19
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
#20
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
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