generation Protocols

Category: RNA Protocols: 5' RACE - Rapid Amplification of cDNA Ends Protocols

Can also use 96-well plates. First Strand cDNA Synthesis, First Size selection, second 2nd strand cDNA synthesis. Bay Area Functional Genomics Consortium, UCSF. - [Read 5' RACE Protocol For Generation of Seq. Tags]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

A simplified system for rapid generation of recombinant adenoviruses. Includes: Generation of Recombinants in Bacterial Cells; Viral Production in 293 or 911 Cells; Preparation of High Titer Viral Stocks. - [Read A Simplified System for Rapid Generation of Recombinant Adenoviruses]

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: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

Shotgun sequencing of a large segment of DNA involves random fragmentation of the target region into smaller segments that are subsequently cloned into a bacteriophage M13 vector. The goal is to create a library of overlapping clones that provide at least fivefold coverage over the entire length of the target fragment. - [Read Generation of a Library of Randomly Overlapping DNA Inserts Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Protocol for the generation of B95-8 EB-Virus. - [Read Generation of B95-8 EB-Virus Protocol]

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

In this method, the nuclease BAL 31 is used to make uni- or bidirectional deletions in a segment of cloned DNA. BAL 31 is a complex enzyme and tends to digest a population of double-stranded DNA targets in an asynchronous fashion, Deletions created by BAL 31 are therefore far more heterogeneous in size than those created by processive enzymes such as exonuclease III. - [Read Generation of Bidirectional Sets of Deletion Mutants by Digestion with BAL 31 Nuclease Protocol]

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for the generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system. Technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. - [Read Generation of Gene Deletions and Gene Replacements in Escherichia coli Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Generation of a growth curve can be useful in evaluating the growth characteristics of a cell line. From a growth curve, the lag time, population doubling time, and saturation density can be determined. - [Read Generation of Growth Curve Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocol for generation of multivirus-specific CTLs. - [Read Generation of Multivirus-Specific CTLs Protocol]

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

The double-stranded DNA of recombinant plasmid, phagemid, or bacteriophage M13 replicative form DNA is digested with two restriction enzymes whose sites of cleavage both lie between one end of the target DNA and the binding site for universal primer. The enzyme that cleaves nearer the target sequence must generate either a blunt end or a recessed 3' terminus; the other enzyme must generate a four-nucleotide protruding 3' terminus. - [Read Generation of Sets of Nested Deletion Mutants with Exonuclease III Protocol]

Category: Fungi Protocols: Neurospora Protocols

Protocol for generation of transformable spheroplasts from mycelia, macroconidia, microconidia and germinating ascospores of Neurospora crassa. - [Read Generation of Transformable Spheroplasts Protocol]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol for the immunoprecipitation (IP) of Homer 1a, injection of virions and in situ hybridization in the spinal cord. Includes: Immunoprecipitation (IP) of Homer1a from spinal cord; Injection of virions in the parenchyma of the spinal dorsal horn in vivo; Generation of cRNA probes; Analysis of DIG-dUTP incorporation; Tissue hybridization. - [Read Immunoprecipitation of Homer 1a, Injection of Virions and In Situ Hybridization in the Spinal Cord]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Describes the basic principles of in situ hybridization and advantages and disadvantages of different methodologies that can be used. Includes: Probe Selection; Probe Generation; Probe Labels; Fixation of Tissue; Hybridization and Washing; Control Procedures. - [Read In situ Hybridization]

Category: Recombinant Protein Protocols: Baculovirus Recombinant Protein

Generation of Recombinant Protein with Baculovirus Infections Protocols and Procedures. Frank Lab. - [Read Insect Cell Culture and Baculovirus Infections]

Category: PCR Protocols: Inverse PCR Protocols

Protocol for inverse PCR & cycle sequencing of P element insertions for STS generation. - [Read Inverse PCR & Cycle Sequencing of P Element Insertions for STS Generation Protocol]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

The latest generation of Promega cell-based assays includes luminescent and fluorescent chemistries to measure markers of cell viability, cytotoxicity and apoptosis, as well as to perform reporter analysis. Using these tools researchers can investigate how cells respond to growth factors, cytokines, hormones, mitogens, radiation, effectors, compound libraries and other signaling molecules. The protocols provided are guidelines for multiplexing cell-based assays & are intended as starting points. - [Read Multiplexing Cell Viability Assays Protocols]

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: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

The protocol consists of a method for the generation of cytoplasmic extracts from mammalian cells (in this case, 293T cells) without the disruption of polyribosomes, the separation of ribosomal components and polyribosomes by sucrose gradient centrifugation, the isolation of mRNA from these fractions, and detection of mRNA by Northern blot analysis. - [Read Northern Blot Analysis of mRNA From Mammalian Polyribosomes Protocol]

Category: Antibody Protocols: Antibody Production Protocols

Protocol for polyclonal antibody production. Very useful for rapid and simple generation of antibodies for western blots, ELISA assays, and immunoprecipitation. Includes: Rabbit Immunization; Initial Preparation; Pre-bleed; Antigen Injection; Monitoring of Titer; Purification of Antibodies. - [Read Polyclonal Antibody Production Protocol]

Category: Peptide Protocols: Antibodies from Peptides Protocols

Preparation Of Peptide-KLH Conjugates For Immunization. Coupling of Peptide to KLH. And generation of Antibodies using peptide-KLH conjugate - Claire Walczak. - [Read Preparation Of Peptide-KLH Conjugates For Immunization]

Category: RNA Protocols: RT-PCR and cDNA synthesis

Protocol for First-strand cDNA Synthesis (PCR template generation with M-MuLV Reverse Transcriptase) - [Read Protocol for First-strand cDNA Synthesis Fermentas]

Category: PCR Protocols: Real-Time PCR Protocols

Protocol uses the Superscript II First-Strand Synthesis system for the generation of cDNA from total RNA.  RNA purified using TRIzol reagent (Invitrogen) or the methods described in Preparation of RNA Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifugation, Preparation of RNA from Paraffin-Embedded Fixed Tissue. - [Read Real-Time RT-PCR: cDNA Synthesis Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses the Superscript II First-Strand Synthesis system for the generation of cDNA from total RNA.  RNA purified using TRIzol reagent (Invitrogen) or the methods described in Preparation of RNA Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifugation, Preparation of RNA from Paraffin-Embedded Fixed Tissue. - [Read Real-Time RT-PCR: cDNA Synthesis Protocol]

Category: Immunology

Removal of CCR5 ligands and induction of pro-resolving lipid mediators by apoptotic neutrophils during resolution. Application of lipid extraction from peritoneal exudates, in tandem with lipid mediator informatics can be used to determine the role of apoptotic neutrophils in the generation of resolution phase lipid mediators. This neutrophil transfer system allows the determination of the direct impact of apoptotic leukocytes in the resolution of inflammation. - [Read Removal of CCR5 Ligands and Induction of Pro-Resolving Lipid Mediators by Apoptotic Neutrophils]

Category: Plant Biology Protocols: Arabidopsis Transformation Protocols

Arabidopsis can be stably transformed using Agrobacterium tumefaciens-mediated transfer of T-DNA. We describe the generation of transgenic plants via root transformation in tissue culture, which can be useful for transforming sterile mutants. - [Read Root Transformation of Arabidopsis Protocol]