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University of British Columbia - UBC

20 ARTICLES PUBLISHED IN JoVE

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Biology

Single Cell Electroporation in vivo within the Intact Developing Brain
D. Sesath Hewapathirane 1,2, Kurt Haas 1,2
1Brain Research Centre, University of British Columbia - UBC, 2Department of Cellular and Physiological Sciences, University of British Columbia - UBC

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

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Biology

Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis
Lacey Samuels 1, Allan DeBono 1, Patricia Lam 1, Miao Wen 1, Reinhard Jetter 1,2, Ljerka Kunst 1
1Department of Botany, University of British Columbia - UBC, 2Department of Chemistry, University of British Columbia - UBC

The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation but is also an important barrier against the entry of pathogenic microorganisms. In this video, we demonstrate the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.

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Biology

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization
Jennifer Y. Kennett 1, Spencer K. Watson 1, Heather Saprunoff 2, Cameron Heryet 3, Wan L. Lam 1
1Department of Cancer Genetics, BC Cancer Research Centre, 2Deeley Research Centre, BC Cancer Agency, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

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Biology

Methylated DNA Immunoprecipitation
Kelsie L. Thu *1,2, Emily A. Vucic *1,3, Jennifer Y. Kennett 1,3, Cameron Heryet 5, Carolyn J. Brown 5, Wan L. Lam 1,2,3, Ian M. Wilson 1,3
1Department of Cancer Genetics and Developmental Biology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Department of Pathology and Laboratory Medicine, University of British Columbia - UBC, 4Photography/Video Production, Multi-Media Services, BC Cancer Agency, 5Department of Medical Genetics, Life Sciences Institute,, University of British Columbia - UBC

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

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Biology

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes
Winco W.H. Wu 1, Lindsay L. Weaver 1, Nelly Panté 1
1Department of Zoology, University of British Columbia - UBC

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

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Biology

Microinjection of Xenopus Laevis Oocytes
Sarah Cohen 1, Shelly Au 1, Nelly Panté 1
1Department of Zoology, University of British Columbia - UBC

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

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JoVE Core

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes
Dee Brink 1, Mary Gilbert 1, Vanessa Auld 1
1Department of Zoology, University of British Columbia - UBC

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

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Biology

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc
Patrick Cafferty 1, Xiaojun Xie 1, Kristen Browne 1, Vanessa J. Auld 1
1Department of Zoology, University of British Columbia - UBC

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

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Biology

Seawater Sampling and Collection
Elena Zaikova 1, Alyse Hawley 1, David A. Walsh 1, Steven J. Hallam 1
1Department of Microbiology and Immunology, University of British Columbia - UBC

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

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Biology

Large Volume (20L+) Filtration of Coastal Seawater Samples
David A. Walsh 1, Elena Zaikova 1, Steven J. Hallam 1
1Department of Microbiology and Immunology, University of British Columbia - UBC

This video documents large volume (≥20 L) filtration of microbial biomass, ranging between 0.22μm and 2.7μm in diameter, from the water column.

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JoVE Core

Small Volume (1-3L) Filtration of Coastal Seawater Samples
David A. Walsh 1, Elena Zaikova 1, Steven J. Hallam 1
1Department of Microbiology and Immunology, University of British Columbia - UBC

This video documents small volume (~1 L) filtration of microbial biomass from the water column.

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Biology

A high-throughput method to globally study the organelle morphology in S. cerevisiae
Shabnam Tavassoli 1, Jesse Tzu-Cheng Chao 1, Christopher Loewen 1
1Department of Cellular and Physiological Sciences, University of British Columbia - UBC

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

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Biology

Identification of protein complexes with quantitative proteomics in S. cerevisiae
Jesse Tzu-Cheng Chao 1, Leonard J. Foster 2, Christopher J. R. Loewen 1
1Department of Cellular and Physiological Sciences, University of British Columbia - UBC, 2Department of Biochemistry and Molecular Biology, University of British Columbia - UBC

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

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Biology

DNA Extraction from 0.22 μM Sterivex Filters and Cesium Chloride Density Gradient Centrifugation
Jody J. Wright 1, Sangwon Lee 1, Elena Zaikova 1, David A. Walsh 1, Steven J. Hallam 1
1Department of Microbiology and Immunology, University of British Columbia - UBC

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 μm Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

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Biology

Large Insert Environmental Genomic Library Production
Marcus Taupp 1, Sangwon Lee 1, Alyse Hawley 1, Jinshu Yang 1, Steven J. Hallam 1
1Department of Microbiology and Immunology, University of British Columbia - UBC

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

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Biology

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments
Sangwon Lee 1, Steven J. Hallam 1
1Department of Microbiology and Immunology, University of British Columbia - UBC

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

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Biology

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Maria Weidner 1, Marcus Taupp 1, Steven J. Hallam 1
1Department of Microbiology and Immunology, University of British Columbia - UBC

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

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Biology

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries
Keith Mewis 1, Marcus Taupp 1, Steven J. Hallam 1
1Microbiology and Immunology, University of British Columbia - UBC

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

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Biology

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses
Larissa A. Pikor *1,2, Katey S. S. Enfield *1,2, Heryet Cameron 3, Wan L. Lam 1,2,4
1Department of Integrative Oncology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency, 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

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Environment

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis
David Colatriano 1, David A. Walsh 1
1Department of Biology, Concordia University

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

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