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BioTechniques

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https://www.readbyqxmd.com/read/28701148/improved-specificity-of-western-blot-following-processing-at-4%C3%A2-c-using-blotcycler
#1
(no author information available yet)
No abstract text is available yet for this article.
July 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28701147/combinational-use-of-lipid-based-reagents-for-efficient-transfection-of-primary-fibroblasts-and-hepatoblasts
#2
Kazuhiro Ishiguro, Osamu Watanabe, Masanao Nakamura, Takeshi Yamamura, Masanobu Matsushita, Hidemi Goto, Yoshiki Hirooka
Commercially available lipid-based transfection reagents are widely used to deliver DNA to cells. However, these lipid-based transfection reagents show poor gene transfer efficiency in primary cells. Here, we demonstrate a simple method to improve gene transfer efficiency in primary fibroblasts and hepatoblasts using a combination of lipid-based transfection reagents. Our data show that combined use of Lipofectamine LTX and FuGENE HD increases the efficiency of gene transfer compared with the use of either reagent alone, and this combination achieves the best result of any pairwise combination of Lipofectamine LTX, FuGENE HD, TransFectin, and Fibroblast Transfection Reagent...
July 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28701146/counting-nuclei-released-from-microcarrier-based-cultures-using-pro-fluorescent-nucleic-acid-stains-and-volumetric-flow-cytometry
#3
Michael Y C He, Steven A Stacker, Ralph Rossi, Michael M Halford
Counting nuclei released from intact cells is a convenient and reliable approach to assess cell density during microcarrier-based culture of adherent cells. However, commonly used methods for counting nuclei, such as crystal violet staining and quantification with a hemocytometer/automated imaging system or a Coulter counter, are imprecise, laborious and, limited in throughput. Here, we describe the use of high-affinity pro-fluorescent nucleic acid stains and volumetric flow cytometry for automated counting of nuclei released from cells attached to microcarriers with improved precision and high sample throughput...
July 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28701145/an-in-vitro-technique-to-identify-the-rna-binding-site-sequences-for-rna-binding-proteins
#4
SunKyung Choi, Chungoo Park, Kyoon Eon Kim, Kee K Kim
RNA-protein interactions play a major role in gene regulation. Although many techniques to analyze RNA-protein interactions have been developed, noteworthy challenges such as determining the RNA sequences that bind RNA-binding proteins (RBPs) remain unsolved. Here, we describe a novel technique using a 4-thio-uridine-incorporated RNA pool to identify the RBP-binding consensus sequences for RBPs produced by in vitro transcription and translation. To confirm the fidelity of this approach, we determined the consensus RBP-binding sequence for RBFOX2, UGC(A/U)(A/U)NU, which is very similar to the known RBFOX2-binding sequence, UGCAUG...
July 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28701144/template-dependent-multiple-displacement-amplification-for-profiling-human-circulating-rna
#5
Weihua Wang, Yi Ren, Yang Lu, Yuan Xu, Seth D Crosby, Adrian M Di Bisceglie, Xiaofeng Fan
Multiple displacement amplification (MDA) is widely used in whole-genome/transcriptome amplification. However, template-independent amplification (TIA) in MDA is a commonly observed phenomenon, particularly when using high concentrations of random hexamer primers and extended incubation times. Here, we demonstrate that the use of random pentamer primers with 5´ ends blocked by a C18 spacer results in MDA solely in a template-dependent manner, a technique we have named tdMDA. Together with an optimized procedure for the removal of residual genomic DNA during RNA extraction, tdMDA was used to profile circulating RNA from 0...
July 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28701143/smrt-gate-a-method-for-validation-of-synthetic-constructs-on-pacific-biosciences-sequencing-platforms
#6
Rosalinda D'Amore, James Johnson, Sam Haldenby, Neil Hall, Margaret Hughes, Ryan Joynson, John G Kenny, Nicola Patron, Christiane Hertz-Fowler, Anthony Hall
Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. However, as new automation methods emerge that enable high-throughput assembly, QC using these techniques has become a major bottleneck...
July 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28701142/injecting-nanoparticles-into-immunotherapy
#7
Sarah Webb
Sarah Webb explores nanoscience strategies for activating T cells in the fight against cancer.
July 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625161/recombinase-polymerase-amplification-rpa-an-isothermal-technique-suitable-for-a-range-of-applications-evaluation-of-rpa-volume-tolerance
#8
(no author information available yet)
No abstract text is available yet for this article.
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625160/blotcycler-consistently-shows-an-increase-in-western-blot-signal-intensity
#9
(no author information available yet)
No abstract text is available yet for this article.
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625159/dna-extraction-for-streamlined-metagenomics-of-diverse-environmental-samples
#10
Clarisse Marotz, Amnon Amir, Greg Humphrey, James Gaffney, Grant Gogul, Rob Knight
A major bottleneck for metagenomic sequencing is rapid and efficient DNA extraction. Here, we compare the extraction efficiencies of three magnetic bead-based platforms (KingFisher, epMotion, and Tecan) to a standardized column-based extraction platform across a variety of sample types, including feces, oral, skin, soil, and water. Replicate sample plates were extracted and prepared for 16S rRNA gene amplicon sequencing in parallel to assess extraction bias and DNA quality. The data demonstrate that any effect of extraction method on sequencing results was small compared with the variability across samples; however, the KingFisher platform produced the largest number of high-quality reads in the shortest amount of time...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625158/a-minimally-invasive-method-for-sampling-human-petrous-bones-from-the-cranial-base-for-ancient-dna-analysis
#11
Kendra A Sirak, Daniel M Fernandes, Olivia Cheronet, Mario Novak, Beatriz Gamarra, Tímea Balassa, Zsolt Bernert, Andrea Cséki, János Dani, József Zsolt Gallina, Gábor Kocsis-Buruzs, Ivett Kővári, Orsolya László, Ildikó Pap, Róbert Patay, Zsolt Petkes, Gergely Szenthe, Tamás Szeniczey, Tamás Hajdu, Ron Pinhasi
Ancient DNA (aDNA) research involves invasive and destructive sampling procedures that are often incompatible with anthropological, anatomical, and bioarcheological analyses requiring intact skeletal remains. The osseous labyrinth inside the petrous bone has been shown to yield higher amounts of endogenous DNA than any other skeletal element; however, accessing this labyrinth in cases of a complete or reconstructed skull involves causing major structural damage to the cranial vault or base. Here, we describe a novel cranial base drilling method (CBDM) for accessing the osseous labyrinth from the cranial base that prevents damaging the surrounding cranial features, making it highly complementary to morphological analyses...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625157/purification-of-single-stranded-dna-by-co-polymerization-with-acrylamide-and-electrophoresis
#12
Tulsi Ram Damase, Andrew D Ellington, Peter B Allen
Single-stranded DNA (ssDNA) oligonucleotides are useful as aptamers, hybridization probes and for emerging applications in DNA nanotechnology. Current methods to purify ssDNA require both a strand-separation step and a separate size-separation step but may still leave double-stranded DNA (dsDNA) impurities in the sample. Here, we use commercially available acrydite DNA primers to immobilize one strand of a PCR product within a polyacrylamide matrix. Electrophoresis moves the non-crosslinked DNA into the gel where the single-stranded product of desired size can be recovered...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625156/heteroduplex-cleavage-assay-for-screening-of-probable-zygosities-resulting-from-crispr-mutations-in-diploid-single-cell-lines
#13
Kyle D Luttgeharm, Kit-Sum Wong, Steve Siembieda
The most common gene editing methods, such as CRISPR, involve random repair of an induced double-stranded DNA break through the non-homologous end joining (NHEJ) repair pathway, resulting in small insertions/deletions. In diploid cells, these mutations can take on one of three zygosities: monoallelic, diallelic heterozygous, or diallelic homozygous. While many advances have been made in CRISPR delivery systems and gene editing efficiency, little work has been done to streamline detection of gene editing events...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625155/applications-of-optical-dna-mapping-in-microbiology
#14
Diana Bogas, Lena Nyberg, Rui Pacheco, Nuno F Azevedo, Jason P Beech, Margarita Gomila, Jorge Lalucat, Célia M Manaia, Olga C Nunes, Jonas O Tegenfeldt, Fredrik Westerlund
Optical mapping (OM) has been used in microbiology for the past 20 years, initially as a technique to facilitate DNA sequence-based studies; however, with decreases in DNA sequencing costs and increases in sequence output from automated sequencing platforms, OM has grown into an important auxiliary tool for genome assembly and comparison. Currently, there are a number of new and exciting applications for OM in the field of microbiology, including investigation of disease outbreaks, identification of specific genes of clinical and/or epidemiological relevance, and the possibility of single-cell analysis when combined with cell-sorting approaches...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625154/expanding-the-genetic-alphabet
#15
Janelle Weaver
Synthetic biologists have engineered a semisynthetic organism that can store increased genetic information using an alphabet consisting of six letters that form three base pairs. Janelle Weaver learns how they did it.
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625153/stories-in-ancient-dna
#16
Nathan Blow
Technology developments are leading to rapid advances in ancient DNA analysis. Nathan Blow talks to researchers peering into the past through ancient DNA.
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528577/single-step-pcr-based-genetic-sex-determination-of-rat-tissues-and-cells
#17
Pramod Dhakal, Michael J Soares
The advent of genome editing strategies has expanded the range of animal models available for gene manipulation and renewed research interest in the rat. Gender is a key variable for in vivo gene function analyses. Here, we present a simple PCR-based method to determine genetic sex in the rat.
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528576/using-non-thermal-irreversible-electroporation-to-create-an-in-vivo-niche-for-exogenous-cell-engraftment
#18
Tammy T Chang, Vivian X Zhou, Boris Rubinsky
The critical shortage of donor organs has spurred investigation of alternative approaches to either generate replacement organs or implant exogenous cells for treatment of end-stage organ failure. Non-thermal irreversible electroporation (NTIRE), which uses brief high electric field pulses to induce irreversible permeabilization of cell membranes, has emerged as a technique for tumor ablation. Here, we introduce a new application for NTIRE that employs in situ cell ablation to create a niche within a solid organ for engraftment of exogenous cells in vivo...
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528575/characterization-of-a-cyclic-olefin-polymer-microcentrifuge-tube
#19
Lloyd Waxman, Rachel L Erwin, Vinod D Vilivalam
Here, we describe the properties of a prototype microcentrifuge tube made from the plastic cyclic olefin polymer (COP). This material has been used in the manufacture of primary containers including syringes and vials for the storage, shipment, and delivery of biotherapeutics, vaccines, and cell therapy products. Its low level of extractable substances and metals along with its glass-like clarity make COP an attractive material for the fabrication of microcentrifuge tubes and other consumable laboratory plasticware where contamination is an important consideration, such as in the storage and analysis of labile proteins, nucleic acids, and metabolites...
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528574/esc-track-a-computer-workflow-for-4-d-segmentation-tracking-lineage-tracing-and-dynamic-context-analysis-of-escs
#20
Laura Fernández-de-Manúel, Covadonga Díaz-Díaz, Daniel Jiménez-Carretero, Miguel Torres, María C Montoya
Embryonic stem cells (ESCs) can be established as permanent cell lines, and their potential to differentiate into adult tissues has led to widespread use for studying the mechanisms and dynamics of stem cell differentiation and exploring strategies for tissue repair. Imaging live ESCs during development is now feasible due to advances in optical imaging and engineering of genetically encoded fluorescent reporters; however, a major limitation is the low spatio-temporal resolution of long-term 3-D imaging required for generational and neighboring reconstructions...
May 1, 2017: BioTechniques
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