A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

JoVE 4316 5/16/2013

Dumizulu L. Tembo¹, Jacqui Montgomery¹, Alister G. Craig², Samuel C. Wassmer³

¹Malawi-Liverpool-Wellcome Trust Clinical Research Programme, ²Liverpool School of Tropical Medicine, ³Department of Microbiology, Division of Medical Parasitology, New York University School of Medicine

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

JoVE 2533 3/31/2011

Nicole L. Jacobs¹, R. Craig Albertson¹, Jason R. Wiles^1,2

¹Department of Biology, Syracuse University, ²Department of Science Teaching, Syracuse University

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Electroeluting DNA Fragments

JoVE 2136 9/05/2010

Ana L. Zarzosa-Álvarez, Antonio Sandoval-Cabrera, Ana L. Torres-Huerta, Rosa Ma. Bermudez-Cruz

Department of Genetics and Molecular Biology , Research and Advanced Studies Center of National Polytechnic Institute

This procedure allows the purification of DNA fragments with high yield.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

JoVE 4356 11/23/2012

Xiuchun Ge, Ping Xu

The Philips Institute of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

JoVE 1959 1/26/2011

Ranjodh Sandhu, Bibo Li

Biological, Geo. & Env. Sciences, Cleveland State University

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

JoVE 3150 9/23/2011

Lauren Liddell^1,2, Glenn Manthey², Nicholas Pannunzio³, Adam Bailis²

¹Irell & Manella Graduate School of Biological Sciences, ²Department of Molecular and Cellular Biology, City of Hope Comprehensive Cancer Center and Beckman Research Institute, ³Department of Biochemistry and Molecular Biology, University of Southern California, Norris Comprehensive Cancer Center

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

JoVE 3104 7/15/2011

Ronald W. Jensen, Jason Rivest, Wei Li, Varalakshmi Vissa

Department of Microbiology, Immunology and Pathology, Colorado State University

Leprosy, caused by Mycobacterium leprae, is still endemic in many places. In order to learn about the spread and mode of transmission of leprosy, it is important to determine which strain of M. leprae has infected a patient. Variable numbers of tandem repeats (VNTR) typing is one such method.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

JoVE 3281 1/09/2013

Mulenga Musapa¹, Taida Kumwenda¹, Mtawa Mkulama¹, Sandra Chishimba¹, Douglas E. Norris², Philip E. Thuma¹, Sungano Mharakurwa¹

¹Malaria Institute at Macha, ²Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Avian Influenza Surveillance with FTA Cards: Field Methods, Biosafety, and Transportation Issues Solved

JoVE 2832 8/02/2011

Robert H.S. Kraus¹, Pim van Hooft¹, Jonas Waldenström², Neus Latorre-Margalef², Ronald C. Ydenberg^1,3, Herbert H.T. Prins¹

¹Resource Ecology Group, Wageningen University, ²Section for Zoonotic Ecology and Epidemiology, School of Natural Sciences, Linnaeus University, ³Centre for Wildlife Ecology, Simon Fraser University

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

DNA Extraction from 0.22 μM Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

JoVE 1352 9/18/2009

Jody J. Wright, Sangwon Lee, Elena Zaikova, David A. Walsh, Steven J. Hallam

Department 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.

Agarose Gel Electrophoresis for the Separation of DNA Fragments

JoVE 3923 4/20/2012

Pei Yun Lee, John Costumbrado, Chih-Yuan Hsu, Yong Hoon Kim

Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

JoVE 247 7/29/2007

Shenyuan Zhang, Michael D. Cahalan

Department of Physiology and Biophysics, University of California, Irvine (UCI)

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

JoVE 50156 2/01/2013

Callie L. Janik^1,2, Timothy K. Starr^1,2

¹Department of Obstetrics, Gynecology & Women's Health, Masonic Cancer Center, University of Minnesota, Minneapolis, ²Department of Genetics, Cell Biology & Development, Center for Genome Engineering, University of Minnesota, Minneapolis

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

JoVE 50059 12/06/2012

Nathaniel T. Remlinger^1,2, Peter D. Wearden^1,3, Thomas W. Gilbert^1,2,3,4

¹McGowan Institute for Regenerative Medicine, ²Department of Bioengineering, University of Pittsburgh, ³Department of Cardiothoracic Surgery, Children's Hospital of Pittsburgh of UPMC, ⁴Department of Surgery, University of Pittsburgh

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

JoVE 4016 11/28/2012

Konstantin Galichanin^1,2, Nooshin Talebizadeh², Per Söderberg²

¹St. Erik's Eye Hospital, Karolinska Institutet, ²Gullstrand lab, Section for Ophthalmology, Department of Neuroscience, Uppsala University

Cataract is the leading cause of blindness in the world. Solar ultraviolet radiation (UVR) is the main risk factor for cataract development. An animal model of far UVR-B induced cataract was developed. In this article we describe methods for investigation of cataract formation: exposure to UVR, quantitative RT-PCR and immunohistochemistry.

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

JoVE 3998 5/22/2012

Todd C. Lorenz

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

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.

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

JoVE 3659 11/17/2011

Krissy Remple¹, Laurel Stone²

¹Department of Molecular and Cell Biology, Life Technologies, ²Life Technologies

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

JoVE 550 12/04/2007

L Cristina Gavrilescu, Richard A Van Etten

Molecular Oncology Research Institute, Tufts University

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

JoVE 2855 9/10/2011

Steven Robbins*¹, Jisha Jacob*¹, Xinxin Lu¹, Mary Ann Moran², Xiaozhen Mou¹

¹Biological Sciences, Kent State University, ²Marine Sciences, University of Georgia (UGA)

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

JoVE 2220 12/21/2010

H. Arthur Jeiranian*, Brandy J. Schalow*, Justin Courcelle

Department of Biology, Portland State University

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

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

JoVE 3130 7/22/2011

John J. Maurer, Margie D. Lee, Ying Cheng, Adriana Pedroso

Department of Population Health, University of Georgia

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.

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

JoVE 4202 5/15/2012

David R. Pawlowski^1,2, Richard J. Karalus^1,2

¹CUBRC, Inc., ²Department of Microbiology and Immunology, State University of New York at Buffalo, School of Medicine and Biomedical Sciences

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

JoVE 3265 2/20/2012

Leland J. Cseke¹, Sharon M. Talley²

¹Department of Biological Sciences, The University of Alabama, Huntsville, ²USDA-APHIS-PPQ, Center for Plant Health Science and Technology

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

JoVE 2897 9/04/2011

Luciana Madeira da Silva¹, Lauren Vandepas¹, Suzy D.C. Bianco^1,2

¹Division of Endocrinology and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, ²Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

JoVE 3136 9/27/2011

Shengyuan Ding, Fu- Ming Zhou

Department of Pharmacology, University of Tennessee College of Medicine

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

JoVE 1569 11/10/2009

Sangwon Lee, Steven J. Hallam

Department 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.

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

JoVE 2101 7/07/2010

Michael L. Beshiri¹, Abul Islam², Dannielle C. DeWaal¹, William F. Richter¹, Jennifer Love³, Nuria Lopez-Bigas², Elizaveta V. Benevolenskaya¹

¹Department of Biochemistry and Molecular Genetics, University of Illinois Chicago - UIC, ²Research Unit on Biomedical Informatics, Universitat Pompeu Fabra, ³Genome Technology Core, Whitehead Institute for Biomedical Research

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

JoVE 50057 1/30/2013

Félix Legendre*^1,2, Neal Cody*¹, Carole Iampietro¹, Julie Bergalet¹, Fabio Alexis Lefebvre^1,2, Gaël Moquin-Beaudry^1,2, Olivia Zhang¹, Xiaofeng Wang¹, Eric Lécuyer^1,2

¹Institut de Recherches Cliniques de Montréal (IRCM), ²Department of Biochemistry, Université de Montréal

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

DNA Stable-Isotope Probing (DNA-SIP)

JoVE 2027 8/02/2010

Eric A. Dunford, Josh D. Neufeld

Department of Biology, University of Waterloo

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

JoVE 2777 7/30/2011

Kassandra L. Guthmueller, Maggie L. Yoder, Andrea M. Holgado

Department of Biological Sciences, Southwestern Oklahoma State University

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

JoVE 3183 10/10/2011

C. Bart Rountree¹, Wei Ding¹, Hein Dang¹, Colleen VanKirk², Gay M. Crooks³

¹Department of Pediatrics and Pharmacology, Pennsylvania State College of Medicine, ²Department of Pharmacology, Pennsylvania State College of Medicine, ³Department of Pediatrics, University of California Los Angeles, School of Medicine

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Competitive Genomic Screens of Barcoded Yeast Libraries

JoVE 2864 8/11/2011

Andrew M. Smith*^1,2, Tanja Durbic*^2,3, Julia Oh*⁴, Malene Urbanus^1,2, Michael Proctor⁵, Lawrence E. Heisler^2,3, Guri Giaever^2,6, Corey Nislow^1,2,3

¹Banting and Best Department of Medical Research and Department of Molecular Genetics, University of Toronto, ²Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, ³Donnelly Sequencing Centre, University of Toronto, ⁴Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, ⁵Stanford Genome Technology Center, Stanford School of Medicine, Stanford University, ⁶Department of Pharmaceutical Sciences, University of Toronto

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Large Insert Environmental Genomic Library Production

JoVE 1387 9/23/2009

Marcus Taupp, Sangwon Lee, Alyse Hawley, Jinshu Yang, Steven J. Hallam

Department 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.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

JoVE 1862 2/25/2010

Maria Weidner, Marcus Taupp, Steven J. Hallam

Department 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.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

JoVE 2390 10/19/2010

Ayala Tovy¹, Benjamin Hofmann², Mark Helm², Serge Ankri¹

¹Faculty of Medicine, Rappaport Institute, Technion - Israel Institute of Technology, ²The Pharmacy and Biochemistry Institute, Johannes Gutenberg University

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

Primer-Free Aptamer Selection Using A Random DNA Library

JoVE 2039 7/26/2010

Weihua Pan¹, Ping Xin¹, Susan Patrick¹, Stacey Dean², Christine Keating², Gary Clawson^3,4

¹Department of Pathology, Hershey Medical Center, Pennsylvania State University, ²Department of Chemistry, Pennsylvania State University, ³Departments of Pathology, and Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, ⁴Materials Research Institute, Pennsylvania State University

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

JoVE 3521 12/16/2011

Matthew L. Skiles¹, Suchit Sahai¹, James O. Blanchette²

¹Biomedical Engineering Program, University of South Carolina, ²Chemical Engineering Department, University of South Carolina

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

JoVE 50225 2/04/2013

Jennifer Patterson, Cameron Mura

Department of Chemistry, University of Virginia

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

JoVE 2910 6/13/2011

Aya D. Pusic*¹, Yelena Y. Grinberg*¹, Heidi M. Mitchell², Richard P. Kraig¹

¹Department of Neurology and Committee on Neurobiology, The University of Chicago Medical Center, ²Department of Neurology, The University of Chicago Medical Center

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

JoVE 4056 11/12/2012

Alla Gagarinova*¹, Mohan Babu*^2,3, Jack Greenblatt^1,2, Andrew Emili^1,2

¹Department of Molecular Genetics, University of Toronto, ²Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, ³Department of Biochemistry, Research and Innovation Centre, University of Regina

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

JoVE 4057 11/12/2012

Mohan Babu^1,2, Olga Kagan¹, Hongbo Guo¹, Jack Greenblatt^1,3, Andrew Emili^1,3

¹Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, ²Deparment of Biochemistry, Research and Innovation Centre, University of Regina, ³Department of Medical Genetics and Microbiology, University of Toronto

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.