A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting
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.
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.
This procedure allows the purification of DNA fragments with high yield.
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.
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
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)
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 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
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.
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.
A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.
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
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.
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.
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.
This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.
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
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
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
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.
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
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
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
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.
A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.
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.
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 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.
Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.
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.
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.
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.
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
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.
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.
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.
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
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
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
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.
We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.
This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.
Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.
Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.
Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.
Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.
1Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University (Medical Sciences), 2Gifted and Talented Students Office, Educational Development Center, Tabriz University (Medical Sciences), 3School of Advanced Biomedical Sciences, Tabriz University (Medical Sciences)
The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.
1Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's, Harvard Medical School, 2Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's Hospital, 3Brigham and Women's Hospital, Harvard Medical School, 4Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital
Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.