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General Laboratory Techniques

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Basic Methods in Cellular and Molecular Biology

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Model Organisms I

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JoVE Biology
 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 Biology

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

1Department of Biological Sciences, Center for Applied Chemical Biology, Youngstown State University


JoVE 51934

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

 JoVE Biology

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

1Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 2Montreal Neurological Institute, 3Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and Department of Pediatrics, University of Ottawa


JoVE 52295

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

 JoVE Biology

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

1Clinical Research Division, Fred Hutchinson Cancer Research Center, 2Division of Hematology, The Children's Hospital of Philadelphia, 3Department of Pathology, The Children's Hospital of Philadelphia


JoVE 52009

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

 JoVE Biology

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

1Institute for Physiology, Pathophysiology, & Toxicology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke, 2Institute for Immunology & Experimental Oncology, Centre for Biomedical Research and Training (ZBAF), University of Witten/Herdecke


JoVE 51857

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

 JoVE Biology

The Soft Agar Colony Formation Assay

1Department of Hematology and Oncology, University of Illinois at Chicago, 2Department of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, 3Jesse Brown Veterans Affairs Medical Center


JoVE 51998

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

 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

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

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


JoVE 51720

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

 JoVE Biology

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

1Harper Cancer Research Institute, 2Microbiology and Immunology, Indiana University School of Medicine, 3Department of Biological Sciences, University of Notre Dame


JoVE 52022

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

 JoVE Biology

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

1Department of Ophthalmology, Inselspital, University of Bern, 2Department of Ophthalmology, University Hospital of Basel, 3Department of Biology, University of Fribourg


JoVE 51909

Herein we demonstrate quantification of retinal de- and regeneration and its impact on visual function using N-methyl-N-nitrosourea in the adult zebrafish. Loss of visual acuity and decreased photoreceptor numbers were followed by proliferation in the inner nuclear layer. Complete morphological and functional regeneration occurred 30 days after the initial treatment.

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