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  JoVE Medicine

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 JoVE Biology

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

1Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles

JoVE 3998

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

 JoVE Bioengineering

Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis

1School of Sport & Exercise Science, University of Northern Colorado, 2Kinesiology Program, Arizona State University, 3Department of Kinesiology, Iowa State University

JoVE 50977

Body segmental inertial properties are required for inverse dynamics modeling. Using an oscillation and reaction board technique, inertial properties of below-knee prostheses were measured. Using direct measures of prosthesis inertia in the inverse dynamics model of the prosthetic leg resulted in lower magnitudes of resultant joint forces and moments.

 JoVE Behavior

The 5-Choice Serial Reaction Time Task: A Task of Attention and Impulse Control for Rodents

1Department of Neuroscience, Oberlin College

JoVE 51574

This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. Test day challenges, which are modifications of the standard task, increase flexibility of the task and can be combined with other manipulations to more fully characterize behavior.

 JoVE Chemistry

Mizoroki-Heck Cross-coupling Reactions Catalyzed by Dichloro{bis[1,1',1''-(phosphinetriyl)tripiperidine]}palladium Under Mild Reaction Conditions

1Institute of Inorganic Chemistry, University of Zürich, 2Institute of Chemistry & Biological Chemistry, Zürich University of Applied Sciences

JoVE 51444

Dichloro{bis[1,1',1''-(phosphinetriyl)tripiperidine]}palladium [(P(NC5H10)3)2Pd(Cl)2] (1) is an easy accessible, cheap, and air stable, but highly active Heck catalyst with an excellent functional group tolerance that efficiently operates under mild reaction conditions to give the coupling products in very high yields.

 Science Education: Essentials of Cognitive Psychology

Measuring Reaction Time and Donders' Method of Subtraction

JoVE Science Education

Source: Laboratory of Jonathan Flombaum—Johns Hopkins University

The ambition of experimental psychology is to characterize the mental events that support the human ability to solve problems, perceive the world, and turn thoughts into words and sentences. But people cannot see or feel those mental events; they cannot be weighed, combined in test tubes, or grown in a dish. Wanting to study mental life, nonetheless, Franciscus Donders, a Dutch ophthalmologist in the early 1800s, came up with a property that he could measure—even back then: he measured the time it took for human subjects to perform simple tasks, reasoning that he could treat those measurements as proxies for the time it takes to complete the unobservable mental operations involved. In fact, Donders went one step further, developing a basic experimental paradigm known as the Method of Subtraction. It simply asks a researcher to design two tasks that are identical in nearly every way, excepting a mental operation hypothesized to be involved in one of the tasks and omitted in the other. The researcher then measures the time it takes to complete each task, and by subtracting the outcomes, he extracts an estimate of the time it takes to execute the one mental operation of interest. In this way, the method allows a researcher

 JoVE Chemistry

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

1Center for Systems Biology, Massachusetts General Hospital

JoVE 50772

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

 JoVE Immunology and Infection

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

1Pneumococcal Research, Murdoch Childrens Research Institute, 2Department of Microbiology & Immunology, The University of Melbourne

JoVE 51208

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

 JoVE Biology

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

1Cellular Networks Proteomics Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health

JoVE 52959

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

 JoVE Chemistry

Preparation and Use of Samarium Diiodide (SmI2) in Organic Synthesis: The Mechanistic Role of HMPA and Ni(II) Salts in the Samarium Barbier Reaction

1Department of Chemistry, Lehigh University

JoVE 4323

A straightforward procedure for the preparation of samarium diiodide (SmI2) in THF is described. The role of two main additives namely hexamethylphosphoramide (HMPA) and Ni(acac)2 in Sm mediated reactions is demonstrated in the Sm-Barbier reaction.

 JoVE Neuroscience

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

1CullenWebb Animal Neurology & Ophthalmology Center, Riverview, NB, 2Department of Clinical Neurosciences, Faculty of Medicine, University of Calgary, 3Department of Comparative Biology and Experimental Medicine, University of Calgary, 4Department of Neuroscience, University of Calgary

JoVE 2138

Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.

 JoVE Biology

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

1Department of Chemistry, Drexel University, 2Mettler-Toledo, 3Chemspider

JoVE 942

The Ugi reaction has proved to be a convenient way to quickly create diverse libraries of compounds. It involves the reaction of an amine, an aldehyde, a carboxylic acid and an isonitrile typically in methanol at room temperature. In this video, we utilize a 48-slot Mettler-Toledo MiniBlock equipped with filtration tubes and a Mettler-Toledo MiniMapper automated liquid handler was used to deliver the reagents and solvent. The parameters of interest were the concentration, the solvent composition and the excess of some of the reagents.

 JoVE Chemistry

Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents

1Faculty of Pharmacy, The University of Sydney, 2School of Science and Health, University of Western Sydney, 3Division of Chemistry and Environmental Science, School of Science and the Environment, Manchester Metropolitan University, 4Nature Publishing Group

JoVE 51740

This protocol describes the use of amide coupling reactions of isonicotinic acid and diaminoalkanes to form bridging ligands suitable for use in the synthesis of multinuclear platinum complexes, which combine aspects of the anticancer drugs BBR3464 and picoplatin.

 Science Education: Basic Methods in Cellular and Molecular Biology

PCR: The Polymerase Chain Reaction

JoVE Science Education

The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling – cyles of temperature changes at fixed time intervals. Using a thermostable DNA polymerase, PCR can create numerous copies of DNA from DNA building blocks called dinucleoside triphosphates or dNTPs. There are three steps in PCR: denaturation, annealing, and elongation. Denaturation is the first step in the cycle and causes the DNA to melt by disrupting hydrogen bonds between the bases resulting in single-stranded DNA. Annealing lowers the temperature enough to allow the binding of oligonucleotide primers to the DNA template. During the elongation step DNA polymerase will synthesize new double-stranded DNA. This video provides an introduction to the PCR procedure. The basic principles of PCR are described as well as a step-by-step procedure for setting up a generalized PCR reaction. The video shows the necessary components for a PCR reaction, includes instruction for primer design, and provides helpful hints for ensuring successful PCR reactions.

 JoVE Chemistry

Conducting Miller-Urey Experiments

1School of Chemistry and Biochemistry, Georgia Institute of Technology, 2Earth-Life Science Institute, Tokyo Institute of Technology, 3Institute for Advanced Study, 4Astromaterials Research and Exploration Science Directorate, NASA Johnson Space Center, 5Goddard Center for Astrobiology, NASA Goddard Space Flight Center, 6Geosciences Research Division, Scripps Institution of Oceanography, University of California at San Diego

JoVE 51039

The Miller-Urey experiment was a pioneering study regarding the abiotic synthesis of organic compounds with possible relevance to the origins of life. Simple gases were introduced into a glass apparatus and subjected to an electric discharge, simulating the effects of lightning in the primordial Earth’s atmosphere-ocean system. The experiment was conducted for one week, after which, the samples collected from it were analyzed for the chemical building blocks of life.

 JoVE Biology

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

1Department of Medicine, Weill Cornell Medical College, 2Institute for Computational Biomedicine, Weill Cornell Medical College, 3Department of Physiology and Biophysics, Weill Cornell Medical College, 4Department of Pathology, University of Michigan

JoVE 52246

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

 JoVE Biology

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

1Diabetes and Islet Biology Group, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney, 2Biomarkers Laboratory, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney

JoVE 52586

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

 JoVE Behavior

Vision Training Methods for Sports Concussion Mitigation and Management

1Neurology and Rehabilitative Medicine, University of Cincinnati, 2Division of Sports Medicine, Department of Orthopaedic Surgery, University of Cincinnati, 3Department of Athletics, University of Cincinnati, 4Department of Neurosurgery, University of Cincinnati, 5College of Education, Criminal Justice, and Human Services, University of Cincinnati, 6Division of Sports Medicine, Cincinnati Children's Hospital Medical Center

JoVE 52648

This paper describes a protocol to conduct, quantitatively monitor, and assess the success of vision training initiated as part of a sports medical management program including intervention for concussion prevention and performance enhancement.

 JoVE Developmental Biology

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

1Developmental and Stem Cell Biology, Hospital for Sick Children, 2Children's Health Research Institute, University of Western Ontario, 3Department of Physiology and Pharmacology, University of Western Ontario, 4Neurosciences and Mental Health, Hospital for Sick Children, 5Department of Paediatrics, University of Western Ontario

JoVE 51526

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

 JoVE Biology

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

1Department of Bioengineering, Rice University

JoVE 52620

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

 JoVE Bioengineering

Sealable Femtoliter Chamber Arrays for Cell-free Biology

1Bredesen Center, University of Tennessee, Knoxville, 2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 3Department of Materials Science and Engineering, University of Tennessee, Knoxville

JoVE 52616

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

 JoVE Immunology and Infection

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

1Emory Vaccine Center at Yerkes National Primate Research Center, Emory University, 2Department of Pathology and Laboratory Medicine, Emory University

JoVE 51506

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

 JoVE Biology

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center

JoVE 51721

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

 JoVE Biology

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

1Department of Biology, California Institute of Technology, 2Department of Bioengineering, California Institute of Technology, 3Synthetic Biology Center, Department of Bioengineering, Massachusetts Institute of Technology, 4School of Physics and Astronomy, University of Minnesota

JoVE 50762

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

 JoVE Biology

FtsZ Polymerization Assays: Simple Protocols and Considerations

1Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen

JoVE 50844

Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

 JoVE Chemistry

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

1Oak Ridge Institute for Science and Education, 2Air Force Research Laboratory, Airbase Technology Division, 3School of Materials Science and Engineering, Clemson University

JoVE 51022

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

 JoVE Chemistry

Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes

1Department of Chemistry, University of Pittsburgh

JoVE 50511

Microwave-assisted intramolecular dehydrogenative Diels-Alder (DA) reactions provide concise access to functionalized cyclopenta[b]naphthalene building blocks. The utility of this methodology is demonstrated by one-step conversion of the dehydrogenative DA cycloadducts into novel solvatochromic fluorescent dyes via Buchwald-Hartwig palladium-catalyzed cross-coupling reactions.

 JoVE Chemistry

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

1Laboratory of Structural Biology, NIEHS, National Institutes of Health

JoVE 50695

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

 JoVE Biology

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

1Department of Infectious Diseases, The Scripps Research Institute, 2Department of Chemistry, City College of New York

JoVE 51309

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

 JoVE Chemistry

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

1Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna

JoVE 51487

Isothermal titration calorimetry measures heat flow released or absorbed in chemical reactions. This method can be used to quantify enzyme-catalysis. In this paper, the protocol for instrumental setup, experiment running, and data analysis is generally described, and applied to the characterization of enzymatic urea hydrolysis by jack bean urease.

 JoVE Biology

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

1Department of Evolutionary Functional Genomics, Evolutionary Biology Center, Uppsala University, 2Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences

JoVE 1205

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

 JoVE Biology

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

1Boston Biomedical Research Institute

JoVE 3891

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

 JoVE Chemistry

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

1Department of Biomedical Engineering, University of Rochester, 2Department of Chemical Engineering, University of Rochester, 3Center for Musculoskeletal Research, University of Rochester Medical Center

JoVE 50890

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

 JoVE Bioengineering

Synthesis of an Intein-mediated Artificial Protein Hydrogel

1Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, 2Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, Texas A&M University, College Station

JoVE 51202

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

 JoVE Biology

Reconstitution Of β-catenin Degradation In Xenopus Egg Extract

1Department of Cell and Developmental Biology and Program in Developmental Biology, Vanderbilt University Medical Center, 2Division of Gastroenterology, Hepatology & Nutrition and Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine

JoVE 51425

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

 JoVE Biology

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

1Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, 2Hillman Cancer Center, University of Pittsburgh Cancer Institute, 3Department of Experimental Therapy, The Netherlands Cancer Institute, 4Department of Human Genetics, University of Pittsburgh School of Public Health

JoVE 4168

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

 JoVE Immunology and Infection

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

1Department of Pathology and Pathogen Biology, Royal Veterinary College, London, 2BIOPHARM, Research Institute of Biopharmacy and Veterinary Drugs

JoVE 52552

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

 JoVE Immunology and Infection

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

1Department of Population Health, University of Georgia

JoVE 3130

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

 JoVE Biology

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

1Department of Microbial Genetics, Interfaculty Institute of Microbiology and Infection Medicine Tübingen (IMIT), Faculty of Science, University of Tübingen

JoVE 52134

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

 JoVE Medicine

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

1Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston

JoVE 51285

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

 JoVE Biology

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

1Endocrine Research Laboratory and Department of Medicine, Royal Victoria Hospital, 2McGill University Health Centre Research Institute

JoVE 50644

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

 JoVE Immunology and Infection

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

1Department of Microbiology, University of Washington, 2Departments of Medicine and Laboratory Medicine, University of Washington, 3U.S Military HIV Research Program, Walter Reed Army Institute of Research, 4Henry M. Jackson Foundation

JoVE 52610

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

 JoVE Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

1Laboratory Program, BC Centre for Excellence in HIV/AIDS

JoVE 2531

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

 JoVE Neuroscience

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

1BioSentinel Inc., Madison, WI

JoVE 51170

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices. Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

 JoVE Chemistry

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

1Department of Chemistry, Louisiana State University

JoVE 50875

Two methods for assigning the α- and ε-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

 JoVE Chemistry

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

1Department of Chemistry, Imperial College London

JoVE 52689

Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru–N-triphos complex with levulinic acid is described.

 JoVE Biology

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic ‘Touch DNA’ Evidence

1Forensic Science Graduate Program, Biochemistry Track, University of Central Florida, 2Department of Chemistry, University of Central Florida, 3National Center for Forensic Science, University of Central Florida

JoVE 52612

Here we describe an optimized and efficient removal strategy for the collection of bio-particles present in ‘touch DNA’ samples, together with an enhanced amplification protocol involving a one-step 5 µl micro-volume lysis/STR amplification, to permit the recovery of short tandem repeat (STR) profiles of the bio-particle donor(s).

 JoVE Chemistry

Metal-free Synthesis of Ynones from Acyl Chlorides and Potassium Alkynyltrifluoroborate Salts

1Faculty of Science, University of Ontario Institute of Technology

JoVE 52401

The goal of this manuscript is to demonstrate a straightforward method for the preparation of ynones from acyl chloride and potassium alkynyltrifluoroborate salt starting materials. The one-pot reaction proceeds rapidly in the presence of boron trichloride without exclusion of air and moisture.

 JoVE Biology

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

1Department of Marine Sciences, University of Georgia (UGA)

JoVE 1086

We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.

 JoVE Environment

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

1Department of Plant Pathology, University of Georgia

JoVE 50712

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

 JoVE Chemistry

Synthesis and Characterization of Functionalized Metal-organic Frameworks

1Department of Chemistry, Northwestern University, 2Department of Chemistry, Warsaw University of Technology, 3Department of Chemistry, Faculty of Science, King Abdulaziz University

JoVE 52094

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

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