generating Protocols

Category: Fungi Protocols: Aspergillus Protocols

The technique makes use of an Escherichia coli strain expressing the redΑßΓ operon under the control of an inducible promoter. This enables the strain to carry out homologous recombination with only 50-60 bp of homologous sequence. The procedure does not require any DNA ligation and is very rapid. It allows a single gene or region on a cosmid to be replaced by a bi-functional selectable marker (having both an E. coli and an A. fumigatus marker). - [Read A Rapid Method for Generating Gene Deletions in Aspergillus fumigatus Protocol]

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

Describes an experimental cross in mice that can be used to define and map induced germ-line mutations that map to a single chromosome. The cross is a modification and extension of a conventional three-generation recessive mutagenesis screen. Includes: The Mutagenesis Breeding Plan; Consomic Strains; Generating Mutations; Generating and Genotyping G2 Females; Genotyping G3 Progeny; Phenotyping G4 Progeny; etc.. - [Read A Targeted Screen to Detect Recessive Mutations that have Quantitative Effects Protocol]

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This calcium phosphate transfection method works best in cell lines that are 1) highly transformed and 2) adherent (Hela, U2OS, SAOS2, AdAH, NPC-KT and obtain from 20% to 100% transfection efficiency depending on the cell line). Works well for transient experiments but precautions should be used in the design and interpretation of experiments based on the discussion below. Also works very well for generating stable cell lines. This method is quite sensitive to the amount of input plasmid. - [Read Calcium Phosphate Transfection Method]

Category: Cell Biology and Cell Culture

Guide for generating hybridomas. Eppendorf - [Read Guide for generating hybridomas]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to use duplication-generating rearrangements in mapping. - [Read How to Use Duplication-Generating Rearrangements in Mapping]

Category: Immunology: T Cell Protocols

Describes generating CTL against some commonly used target antigens. Two methods for the quantitation of CTL activity are described based on the two pathways used bt CTL to kill target cells. In one pathway, they release lytic granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger apoptosis via Fas/Fas ligand interactions. - [Read Induction and Measurement of Cytotoxic T Lymphocyte Activity Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols

Presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen-presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. - [Read Isolation of Dendritic Cells Protocol]

Category: PCR Protocols: PCR Cloning Protocols

The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments.  This protocol describes the strategies for generating and manipulating suitable ends on the PCR fragments. - [Read Molecular Cloning of PCR Products Protocol]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Describes the steps in detail to isolate and expand neural stem cells in the form of neurospheres from tissue dissections of the post-natal mouse brain. Procedures for the long term passage of neurospheres and the cryopreservation of neurospheres are also provided. In addition to the guidelines and tips for generating neurosphere cultures, we describe the method to prepare neurospheres for analysis by light microscopy. - [Read Neural Stem Cell Culture: Neurosphere Generation, Microscopical Analysis and Cryopreservation]

Category: Viral Manipulation Protocols: Phage Protocols

Protocol describes a method for generating isolated plaques from a stock of bacteriophage lambda. Each plaque derives from infection of a single bacterium by a single bacteriophage particle. Because each plaque contains the progeny of a single virus particle, the bacteriophages derived from a single plaque are essentially genetically identical to one another. - [Read Plating Bacteriophage Lambda Protocol]

Category: Stem Cell Protocols: Stem Cell Injection Protocols

Injection of ES cells into the cavity of blastocysts is one of the main methods for generating chimeras. This protocol describes the preparation of ES cells for injection. - [Read Preparation of Embryonic Stem (ES) Cells for Injection Protocol]