describes Protocols

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes the DNA content of bacteriophage lambda stocks can be easily and rapidly estimated. - [Read Assaying the DNA Content of Bacteriophage lambda Stocks and Lysates by Gel Electrophoresis Protocol]

Category: Immunology: B Cell Protocols

Describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. - [Read Assays for B Lymphocyte Function Protocols]

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for producing diploid embryo-tetraploid embryo chimeras. It requires the timed combination of four-cell-stage tetraploid embryo production and the procedure for diploid embryo-diploid embryo aggregation. The resulting chimeras are useful for phenotypic analysis when an induced mutation has an extraembryonic phenotype. - [Read Assembling Aggregates between Diploid and Tetraploid Embryos Protocol]

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for assembling aggregates between diploid embryos. If embryos from a heterozygous mutant intercross are aggregated with wild-type embryos, the resulting chimeras can be used for analyzing mutant phenotypes. - [Read Assembling Aggregates between Diploid Embryos Protocol]

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for assembling aggregates between ES cells and diploid embryos. The resulting chimeras are useful for separating certain extraembryonic phenotypes from phenotypes in the embryo proper, since the diploid embryo contributes to all parts of the conceptus, but the ES cell component does not contribute to the trophoblast or yolk sac endoderm. - [Read Assembling Aggregates between Embryonic Stem (ES) Cells and Diploid Embryos Protocol]

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for producing ES cell-tetraploid embryo chimeras. It requires the timed combination of four-cell-stage tetraploid embryo production and the procedure for ES cell-diploid embryo aggregation in which diploid embryos are replaced with tetraploid embryos. The resulting chimeras can be used to analyze the embryonic versus extraembryonic phenotype of a mutation. - [Read Assembling Aggregates between Embryonic Stem (ES) Cells and Tetraploid Embryos Protocol]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

This unit describes how to detect and recognize bacterial, fungal, or mycoplasma contamination and how to verify the presence of mycoplasma. Strategies are described for dealing with culture contamination, if necessary. - [Read Assessing and Controlling Microbial Contamination in Cell Cultures Protocol]

Category: DNA Protocols: DNA Sequencing Protocols

Protocol describes fixation of a sequencing gel and processing in preparation for autoradiography. - [Read Autoradiography and Reading of Sequencing Gels Protocol]

Category: Cell Culture Protocols: Sterile Technique Protocols

Cultured mammalian cells are used extensively in cell biology studies; it requires a number of special skills in order to be able to preserve the structure, function, behavior and biology of the cells. This unit describes the basic skills required to maintain and preserve cell cultures: aseptic technique, medium characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. - [Read Basic Techniques for Mammalian Cell Tissue Culture Protocol]

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

Cultured mammalian cells are used extensively in cell biology studies; it requires a number of special skills in order to be able to preserve the structure, function, behavior and biology of the cells. This unit describes the basic skills required to maintain and preserve cell cultures: aseptic technique, medium characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. - [Read Basic Techniques for Mammalian Cell Tissue Culture Protocol]

Category: Mouse Protocols

Protocol describes the strongest way for lineage tracing on sections by using biotin dextran amine. - [Read BDA Mouse Lineage Tracing on Sections Protocol]

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes a stepwise procedure to prepare nucleic acids encapsulated in a polyethylene glycol (PEG)-shielded nanolipoparticle (NLP) that contain a bioresponsive lipid and ligand. This process provides several advantages for systemic gene delivery. The in vivo circulation time is extended. Also, low pH-sensitive lipids enhance DNA unpacking and endosomal escape. Finally, ligands inserted into the NLP surface can target gene delivery to specific tissues or cells in vivo. - [Read Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol]

Category: Mouse Protocols: Mouse Surgical Procedures Protocols

Protocol describes a method for collection of blood from the tail of a mouse. The blood can then be used for analysis. - [Read Blood Collection by Tail Bleeding Protocol]

Category: Stem Cell Protocols: Stem Cell Injection Protocols

Injection needles require sharp tips that allow the blastocyst to be penetrated easily. This protocol describes a method of creating sharp injection needle tips. - [Read Breaking the Tips of Embryonic Stem (ES) Cell Injection Needles Protocol]

Category: Buffers Media, and Solutions: Buffer Theory

This page describes simple acidic and alkaline buffer solutions and explains how they work. - [Read Buffer Solutions and How they work...]

Category: Cell Biology and Cell Culture

OptiPrep Application sheet C14 describes procedures for determining the density and sedimenting properties of any cell (of any size or density) using either a continuous or discontinuous gradient of iodixanol. This Application Sheet describes procedures aimed at isolating specifically a relatively low-density cell fraction from any tissue. - [Read C25 Isolation from spleen, thymus, pancreas, alveolar tissue and other tissues]

Category: Microinjection Protocols: Microinjection Equipment

This protocol describes an easy method for calibrating micropipette tips that have been pulled in the laboratory. It is essential to estimate the internal diameter of the pulled micropipette tip when adjusting parameters for a new puller or new type of glass tubing. A tip diameter of ~0.3 µm is optimal for the microinjection of mammalian cells in culture (e.g., CHO, PtK1, and COS-7). A 10% increase in diameter increases the delivery rate by more than 30% and can cause cell damage. - [Read Calibration of Micropipette Tips Protocols]

Category: Xenopus Protocols

Protocol describes the use of a basic water-based dye to stain for acid mucosubstances and acetic mucins located on the cartilage of fixed embryos, permitting the examination of cartilage formation. - [Read Cartilage Staining of Xenopus tropicalis Protocol]

Category: PCR Protocols: Real-Time PCR Protocols

Protocol describes the real-time PCR using RNA purified from laser-captured tissues Laser Capture Microdissection (LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cells. - [Read cDNA Synthesis and Real-Time PCR Using RNA from Laser-Captured Cells Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Sorting Protocols

The combination of prospective identification/isolation of bone marrow progenitors and quantitative RT-PCR is a powerful tool to understand the molecular mechanism underlying hematopoiesis. Describes the standard procedures of the murine myeloid progenitor staining for fluorescence activated cells sorting (FACS) and RNA purification methods. - [Read Cell Staining for Sorting of Hematopoietic Stem Cells (HSC) and Myeloid Progenitors]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols

protocol describes a method for the synchronization of cell populations using centrifugal elutriation. The method relies on the fact that cell size increases linearly as cells proceed through the cell cycle. Cells of similar size (and cell cycle phase) are eluted stepwise from the cell chamber, with the smallest size (those in early G1) being eluted first. Using this procedure, it is possible to obtain relatively pure populations of cells at various points in G1, S, and G2/M. - [Read Cell Synchronization Using Centrifugal Elutriation Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Cycle Analysis Protocols

This protocol describes a method for the synchronization of cell populations using centrifugal elutriation. The method relies on the fact that cell size increases linearly as cells proceed through the cell cycle. Cells of similar size (and cell cycle phase) are eluted stepwise from the cell chamber, with the smallest size (those in early G1) being eluted first. Using this procedure, it is possible to obtain relatively pure populations of cells at various points in G1, S, and G2/M. - [Read Cell Synchronization Using Centrifugal Elutriation Protocol]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

This protocol describes how to use DIG Chem-Link to directly label any DNA [e.g. plasmids, PCR products, cDNA prepared from mRNA] or RNA (e.g. total RNA, poly(A)+ mMRNA). The DIG Chem-Link or Biotin Chem-Link may also be used to label oligonucleotides. Includes: Required Purity of DIG Chem-Link Templates; Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Key Product Required for Direct Labeling of DNA or RNA; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Protocol describes the use of chromatin immunoprecipitation technology (ChIP) to analyze interactions of proteins or protein complexes with DNA in vivo. In this approach, the material is fixed with formaldehyde to preserve DNA-protein and protein-protein associations, the cells are lysed, and the chromatin is cut and solubilized. The chromatin suspension is immunoprecipitated with an antibody against the protein(s) of interest, and the coimmunoprecipitated DNA fragments are analyzed. - [Read Chromatin Immunoprecipitation (ChIP) of Protein Complexes Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol describes three standard methods to construct bacteriophage M13 recombinants: (1) ligating insert DNA to a linearized vector, prepared by cleavage of M13 RF with a single restriction enzyme; (2) using alkaline phosphatase to suppress self-ligation of the linearized vector, and (3) using M13 RF cleaved with two restriction enzymes for directional cloning. - [Read Cloning into Bacteriophage M13 Vectors Protocol]