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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
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
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
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
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
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
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
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
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
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
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
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
(no author information available yet)
No abstract text is available yet for this article.
February 2016: BioTechniques
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
Weiliang Huang, Wayne A Johnston, Mikael Boden, Elizabeth M J Gillam
Directed evolution has greatly facilitated protein engineering and provided new insights into protein structure-function relationships. DNA shuffling using restriction enzymes is a particularly simple and cost-effective means of recombinatorial evolution that is well within the capability of most molecular biologists, but tools for the design and analysis of such experiments are limited. Here we introduce a suite of freely available online tools to make the construction and analysis of chimeric libraries readily accessible to the novice...
February 2016: BioTechniques
Anita Mäki, Antti J Rissanen, Marja Tiirola
Sample barcoding facilitates the analysis of tens or even hundreds of samples in a single next-generation sequencing (NGS) run, but more efficient methods are needed for high-throughput barcoding and size-trimming of long PCR products. Here we present a two-step PCR approach for barcoding followed by pool shearing, adapter ligation, and 5' end selection for trimming sets of DNA templates of any size. Our new trimming method offers clear benefits for phylogenetic studies, since targeting exactly the same region maximizes the alignment and enables the use of operational taxonomic unit (OTU)-based algorithms...
February 2016: BioTechniques
Simeon Santourlidis, Foued Ghanjati, Agnes Beermann, Thomas Hermanns, Cédric Poyet
Sensitive, accurate, and reliable measurements of tumor cell-specific DNA methylation changes are of fundamental importance in cancer diagnosis, prognosis, and monitoring. Real-time methylation-specific PCR (MSP) using intercalating dyes is an established method of choice for this purpose. Here we present a simple but crucial adaptation of this widely applied method that overcomes a major obstacle: genetic abnormalities in the DNA samples, such as aneuploidy or copy number variations, that could result in inaccurate results due to improper normalization if the copy numbers of the target and reference sequences are not the same...
February 2016: BioTechniques
Maria Zlobina, Ondrej Sedo, Ming-Yuan Chou, Lucia Slepankova, Peter J Lukavsky
Sequence-specific RNA recognition by RNA-binding proteins plays a crucial role in the post-translational regulation of gene expression. Biophysical and biochemical studies help to unravel the principles of sequence-specific RNA recognition, but the methods used require large amounts of single-stranded RNA (ssRNA). Here we present a fast and robust method for large-scale preparation and purification of short ssRNA oligonucleotides for biochemical, biophysical, and structural studies. We designed an efficiently folding, self-cleaving hammerhead (HH) ribozyme to prepare ssRNA oligonucleotides...
February 2016: BioTechniques
Ryan M Sheridan, David L Bentley
Manipulation of protein stability with ligand-regulated degron fusions is a powerful method for investigating gene function. We developed a selectable cassette for easy C-terminal tagging of endogenous human proteins with the E. coli dihydrofolate reductase (eDHFR) degron using CRISPR/Cas9 genome editing. This cassette permits high-efficiency recovery of correct integration events using an in-frame self-cleaving 2A peptide and the puromycin resistance gene. PCR amplified donor eDHFR cassette fragments with 100 bases of homology on each end are integrated by homology-directed repair (HDR) of guide RNA (gRNA)-targeted double-stranded DNA breaks at the 3' ends of open reading frames (ORFs)...
February 2016: BioTechniques
Olga A Zlobovskaya, Tatiana F Sergeeva, Marina V Shirmanova, Varvara V Dudenkova, George V Sharonov, Elena V Zagaynova, Konstantin A Lukyanov
Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new genetically encoded sensors for caspase-3 activity possessing the most red-shifted spectra to date. These consist of Förster resonance energy transfer (FRET) pairs in which a far-red fluorescent protein (mKate2 or eqFP650) is connected to the infrared fluorescent protein iRFP through a linker containing the DEVD caspase-3 cleavage site...
February 2016: BioTechniques
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