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RNA, Bacterial: Ribonucleic acid in bacteria having regulatory and catalytic roles as well as involvement in protein synthesis.

RNAi in C. elegans

JoVE 5105

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target gene. In the nematode, C. elegans, RNAi is particularly easy and effective because it can be delivered simply by feeding the worms bacteria that express double stranded RNA (dsRNA) that is complementary to a gene of interest. First, this video will introduce the concept of RNA interference and explain how it causes targeted gene knockdown. Then, we will demonstrate a protocol for using RNAi in C. elegans, which includes preparation of the bacteria and RNAi worm plates, culturing of the worms, and how to assess the effects of RNAi on the worms. RNAi is frequently used to perform reverse genetic screens in order to reveal which genes are important to carry out specific biological processes. Furthermore, automated reverse genetic screens allow for the efficient knockdown and analysis of a large collection of genes. Lastly, RNAi is often used to study the development of C. elegans. Since its discovery, scientists have used RNAi to make tremendous progress on the understanding of many biological phenomena.


 Biology I

Plasmid Purification

JoVE 5062

Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study. In this video, a step-by-step generalized procedure is described for how to perform plasmid purification. Plasmid purification includes three basic steps: growth of the bacterial culture, harvesting and lysis of the bacteria, and purification of the plasmid DNA. The video contains an explanation where the plasmid can be found in each step of the protocol and to quantitatively and qualitatively analyze plasmid DNA with a spectrophotometer and/or gel electrophoresis. There are different types of plasmid purification methods available, which are geared toward desired yield, plasmid copy number, and bacterial culture volume.


 Basic Methods in Cellular and Molecular Biology

Community DNA Extraction from Bacterial Colonies

JoVE 10218

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Luisa Ikner

Traditional methods of analysis for microbial communities within soils have usually involved either cultural assays utilizing dilution and plating methodology on selective and differential media or direct count assays. Direct counts offer information about the total number of bacteria present, but give no information about the number or diversity of populations present within the community. Plate counts allow enumeration of total cultural or selected cultural populations, and hence provide information on the different populations present. However, since less than 1% of soil bacteria are readily culturable, cultural information offers only a piece of the picture. The actual fraction of the community that can be cultured depends on the medium chosen for cultural counts. Any single medium will select for the populations that are best suited to that particular medium. In recent years, the advantages of studying community DNA extracted from soil samples have become apparent. This nonculture-based approach is thought to be more representative of the actual community present than culture-based approaches. In addition to providing information about the types of populations present, this


 Environmental Microbiology

Synthetic Biology

JoVE 5792

This video presents synthetic biology and its role in bioengineering. Synthetic biology refers to the methods used to genetically modify organisms in order to make them capable of producing large quantities of a product. This product could be a protein that the cell already makes, or a new protein that has been encoded in a newly-inserted DNA sequence.

Here, we discuss how an organism's genetic material is modified using transformation or transfection. Then, the process is shown in the laboratory, and the applications of the technique discussed.


 Bioengineering

Detection of Bacteriophages in Environmental Samples

JoVE 10190

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Alex Wassimi

Viruses are a unique group of biological entities that infect both eukaryotic and prokaryotic organisms. They are obligate parasites that have no metabolic capacity, and in order to replicate, rely on host metabolism to produce viral parts that self-assemble inside host cells. Viruses are ultramicroscopic—too small to be viewed with the light microscope, visible only with the greater resolution of the electron microscope. A viral particle consists of a nucleic acid genome, either DNA or RNA, surrounded by a protein coat, known as a capsid, composed of protein subunits or capsomers. In some more complex viruses, the capsid is surrounded by an additional lipid envelope, and some have spike-like surface appendages or tails. Viruses that infect the intestinal tract of humans and animals are known as enteric viruses. They are excreted in feces and can be isolated from domestic wastewater. Viruses which infect bacteria are known as bacteriophages, and those which infect coliform bacteria are called coliphages (Figure 1). The phages of coliform bacteria are found anywhere coliform bacteria are found.


 Environmental Microbiology

Genome Editing

JoVE 5554

A well-established technique for modifying specific sequences in the genome is gene targeting by homologous recombination, but this method can be laborious and only works in certain organisms. Recent advances have led to the development of “genome editing”, which works by inducing double-strand breaks in DNA using engineered nuclease enzymes guided to target genomic sites by either proteins or RNAs that recognize specific sequences. When a cell attempts to repair this damage, mutations can be introduced into the targeted DNA region. In this video, JoVE explains the principles behind genome editing, emphasizing how this technique relates to DNA repair mechanisms. Then, three major genome editing methods—zinc finger nucleases, TALENs, and the CRISPR-Cas9 system—are reviewed, followed by a protocol for using CRISPR to create targeted genetic changes in mammalian cells. Finally, we discuss some current research that applies genome editing to alter the genetic material in model organisms or cultured cells.


 Genetics

Quantifying Environmental Microorganisms and Viruses Using qPCR

JoVE 10186

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz

Quantitative polymerase chain reaction (qPCR), also known as real-time PCR, is a widely-used molecular technique for enumerating microorganisms in the environment. Prior to this approach, quantifying microorganisms was limited largely to classical culture-based techniques. However, the culturing of microbes from environmental samples can be particularly challenging, and it is generally held that as few as 1 to 10% of the microorganisms present within environmental samples are detectable using these techniques. The advent of qPCR in environmental microbiology research has therefore advanced the field greatly by allowing for more accurate determination of concentrations of microorganisms such as disease-causing pathogens in environmental samples. However, an important limitation of qPCR as an applied microbiological technique is that living, viable populations cannot be differentiated from inactive or non-living populations. This video demonstrates the use of qPCR to detect pepper mild mottle virus from an environmental water sample.


 Environmental Microbiology

Genetic Screens

JoVE 5542

Genetic screens are critical tools for defining gene function and understanding gene interactions. Screens typically involve mutating genes and then assessing the affected organisms for phenotypes of interest. The process can be “forward”, where mutations are generated randomly to identify unknown genes responsible for the phenotypes, or it can be “reverse”, where specific genes are targeted for mutation to observe what phenotypes are produced.Here, JoVE reviews various types of genetic screens, including those that depend on either loss-of-function or gain-of-function mutations, which respectively decrease or increase the activity of genes. We then explore general protocols for forward and reverse screens in a popular model organism, the nematode worm. Finally, we highlight how screens are applied in research today, for example to better understand gene interactions that may contribute to neurodegenerative diseases.


 Genetics

Electrophoretic Mobility Shift Assay (EMSA)

JoVE 5694

The electrophoretic mobility shift assay (EMSA) is a biochemical procedure used to elucidate binding between proteins and nucleic acids. In this assay a radiolabeled nucleic acid and test protein are mixed. Binding is determined via gel electrophoresis which separates components based on mass, charge, and conformation.

This video shows the concepts of EMSA and a general procedure, including gel and protein preparation, binding, electrophoresis, and detection. Applications covered in this video include the analysis of chromatin-remodeling enzymes, a modified EMSA that incorporates biontinylation, and the study of binding sites of bacterial response regulators. EMSA, the electrophoretic mobility shift assay, also known as the gel shift assay, is a versatile and sensitive biochemical procedure. EMSA elucidates binding between proteins and nucleic acids by detecting a shift in bands in gel electrophoresis. This video describes the principles of EMSA, provides a general procedure, and discusses some applications. DNA replication, transcription, and repair, as well as RNA processing are all critical biochemical processes. They all involve binding between proteins and nucleic acids. Many serious diseases and disorders are associated with modifications in this


 Biochemistry

C. elegans Maintenance

JoVE 5104

Ceanorhabditis elegans has been, and is still, used to great success as a model organism for studying a variety of developmental, genetic, molecular and even physical phenomena. In order to use C. elegans to its full potential, proper care and attention to the basic maintenance of this powerful organism is essential.

In this video you will learn the basic housing and feeding requirements of C. elegans, how to correctly handle and manipulate worms using a worm pick and how to freeze and recover important worm stocks. Towards the end of the video we will visit a few applications of modifying the housing, feeding and manipulation of these important animals.


 Biology I

Photometric Protein Determination

JoVE 5688

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more a sample contains light-absorbing substances, the less the light will transmit through it. Since the relationship between concentration and absorption is linear, this phenomenon can be used to measure the concentration in samples where it is unknown. This video describes the basics of photometric protein determination and introduces the Bradford Assay and the Lowry Method. The procedure in the video will cover a typical Bradford assay. Applications covered include direct measurement of very small volumes of nucleic acids to characterize concentration and purity, determination of coupling efficiency of a biomimetic material, and another variation of photometric protein determination using Remazol dye. Determining the concentration of a protein in samples is a fundamental step in many biochemical assays. Photometric determination can be done with small sample sizes. The more a sample contains light-absorbing substances, the less the light will transmit through it. This provides a quantitative measurement of the absorbing substances. These concepts are so fundamental to science that the articles that introduced two of the techniques are in the three most cited papers of


 Biochemistry

Invertebrate Lifespan Quantification

JoVE 5338

Many animals naturally stop growing upon reaching adulthood, after which they undergo aging or "senescence" until dying. The amount of time between an organism\'s birth and death is called its lifespan, which can be influenced by various biological and environmental factors. By exposing organisms to different growth conditions, scientists can better understand the factors affecting lifespan. Flies and worms are ideal organisms to perform such experiments, given their short generation time and simple culture requirements. This video provides a brief overview of the factors affecting aging, and goes on to describe basic protocols for invertebrate lifespan quantification experiments. Finally, three research applications of lifespan quantification will be discussed. These experiments explore the effects of diverse factors, such as temperature, drugs, pathogens, and diet, on lifespan.


 Developmental Biology

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

1Center for Oral Biology, University of Rochester Medical Center, 2State Key Laboratory of Oral Diseases, Sichuan University, 3Department of General Medicine, Glostrup Hospital, Glostrup, Denmark, 4Department of Microbiology and Immunology, University of Rochester Medical Center

JoVE 2512


 Immunology and Infection

CRISPR Guide RNA Cloning for Mammalian Systems

1Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Genetics, Harvard Medical School, 3Department of Pathology, Massachusetts General Hospital, 4Institute for Medical Engineering & Science, Massachusetts Institute of Technology, 5Synthetic Biology Center, Massachusetts Institute of Technology, 6Department of Biological Engineering, Massachusetts Institute of Technology, 7Broad Institute

JoVE 57998


 Genetics

Detecting Environmental Microorganisms with the Polymerase Chain Reaction and Gel Electrophoresis

JoVE 10081

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz

Polymerase chain reaction (PCR) is a technique used to detect microorganisms that are present in soil, water, and atmospheric environments. By amplifying specific sections of DNA, PCR can facilitate the detection and identification of target microorganisms down to the species, strain, and serovar/pathovar level. The technique can also be utilized to characterize entire communities of microorganisms in samples. The culturing of microorganisms in the laboratory using specialized growth media is a long-established technique and remains in use for the detection of microorganisms in environmental samples. Many microbes in the natural environment, while alive, maintain low levels of metabolic activity and/or doubling times and are thus referred to as viable but non-culturable (VBNC) organisms. The use of culture-based techniques alone cannot detect these microbes and, therefore, does not provide a thorough assessment of microbial populations in samples. The use of PCR allows for the detection of culturable microbes, VBNC organisms, and those that are no longer alive or active, as the amplification of genetic sequences does not generally require the pre-enrichment of microorga


 Environmental Microbiology

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

1Infectious Diseases Group, Genome Institute of Singapore, 2Department of Chemical and Biomolecular Engineering, National University of Singapore, 3Division of Infectious Diseases, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore

Video Coming Soon

JoVE 58829


 JoVE In-Press

Ultraviolet-Visible (UV-Vis) Spectroscopy

JoVE 10204

Source: Laboratory of Dr. B. Jill Venton - University of Virginia

Ultraviolet-visible (UV-Vis) spectroscopy is one of the most popular analytical techniques because it is very versatile and able to detect nearly every molecule. With UV-Vis spectroscopy, the UV-Vis light is passed through a sample and the transmittance of light by a sample is measured. From the transmittance (T), the absorbance can be calculated as A=-log (T). An absorbance spectrum is obtained that shows the absorbance of a compound at different wavelengths. The amount of absorbance at any wavelength is due to the chemical structure of the molecule. UV-Vis can be used in a qualitative manner, to identify functional groups or confirm the identity of a compound by matching the absorbance spectrum. It can also be used in a quantitative manner, as concentration of the analyte is related to the absorbance using Beer's Law. UV-Vis spectroscopy is used to quantify the amount of DNA or protein in a sample, for water analysis, and as a detector for many types of chromatography. Kinetics of chemical reactions are also measured with UV-Vis spectroscopy by taking repeated UV-Vis measurements over time. UV-Vis measurements are generally taken with a spectrophotometer. UV-Vis is also a very popular detector for other analytical tech


 Analytical Chemistry

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

1Department of Biomedical Sciences and Pathobiology, Virginia Maryland College of Veterinary Medicine, Virginia Tech, 2Department of Biomedical Engineering, Cornell University, 3School of Electrical and Computer Engineering, Cornell University, 4Department of Biomedical Engineering, Duke University

JoVE 54033


 Immunology and Infection

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

1Focal Area Infection Biology, Biozentrum, University of Basel, 2Centre d’Immunologie de Marseille-Luminy, Université de la Méditérannée UM2, INSERM U1104 CNRS UM7280, 3Departmento de Microbiologìa and Instituto de Salud Tropical, Universidad de Navarra, 4BioDataAnalysis GmbH

JoVE 54263


 Immunology and Infection

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