numbers Protocols

Category: Cell Culture Protocols

Protocol describes a method for estimation of mammalian cell number in a defined volume of medium using a hemocytometer. Automated methods using cell-counting devices such as those produced by Coulter are desirable when large numbers of individual samples are to be counted. - [Read Estimation of Cell Number by Hemocytometry Counting Protocol]

Category: Immunology: T Cell Protocols

Describes methods for labeling high or low numbers of lymphocytes with CSFE. Protocols are provided to use CSFE-labeled cells in cell transfer studies or as cells to be cultured in vitro. Detailed guidelines for positioning of CSFE-labeled lymphocytes in lymphoid organs or other tissues are included for those wishing to use this approach to study lymphocyte migration. - [Read Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation]

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: Animal Research Techniques

Minimization of Animal Numbers Used to Obtain Valid Results. - [Read John Hopkins Animals Research Use and Care]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol used chiefly to generate large stocks of double-stranded DNA of strains of M13 that are routinely used as cloning vectors. Large amounts of single-stranded DNA of an individual recombinant may occasionally be needed for specific purposes, e.g., to generate many preparations of a particular radiolabeled probe or to construct large numbers of site-directed mutants. - [Read Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

Simple protocol is used to extract DNA from small numbers of cultured cells and from fragments of soft or bony tissues.  The method is used chiefly to genotype transgenic and knockout mice.  Each 6-10-mm snippet of mouse tail yields 50-100 µg of DNA that can be used in dot or slot blotting to detect a transgene of interest, in Southern hybridization to detect DNA fragments that are <20 kb in size, and as a template in PCRs. - [Read Preparation of Genomic DNA from Mouse Tails and Other Small Samples Protocol]

Category: PCR Protocols: Quantitative PCR Protocols

Protocol that uses the polymerase chain reaction (PCR) to quantitate the numbers of a particular DNA sequence, from 1 to 20,000 molecules per sample.  In addition, it helps assess the presence of contaminating sequences, which can seriously affect the outcome of the procedure. - [Read Quantitation of Rare DNAs by PCR Protocol]

Category: Fungi Protocols: Neurospora Protocols

Protocol for a rapid method for making large numbers of crosses. - [Read Rapid Method for Making Large Numbers of Crosses Protocol]

Category: Nucleic Acid Detection Protocols

Restriction landmark genomic scanning (RLGS) is a method to detect large numbers of restriction landmarks in a single experiment.  It is based on the concept that restriction enzyme sites can serve as landmarks throughout a genome.  RLGS uses direct end-labeling of the genomic DNA digested with a rare-cutting restriction enzyme and high-resolution two-dimensional electrophoresis. - [Read Restriction Landmark Genomic Scanning Protocol]

Category: Bacterial Protocols

Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the filters are processed for hybridization to an appropriate radiolabeled probe while the original (master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their original positions. - [Read Screening Bacterial Colonies by Hybridization: Intermediate Numbers Protocol]

Category: Bacterial Protocols

This procedure is used to plate, replicate, and subsequently screen large numbers of bacterial colonies (up to 2 x 104 colonies per 150-mm plate or 104 colonies per 90-mm plate). - [Read Screening Bacterial Colonies by Hybridization: Large Numbers Protocol]

Category: Bacterial Protocols

Protocol used to screen a small number of bacterial colonies (<200) that are dispersed over several agar plates and are to be screened by hybridization to the same radiolabeled probe. The colonies are gridded onto a master plate and onto a nitrocellulose or nylon filter laid on the surface of a second agar plate. - [Read Screening Bacterial Colonies by Hybridization: Small Numbers Protocol]

Category: Bacteria Transformation Protocols

Protocol describes a method for transforming Agrobacterium with plasmid DNA using electroporation in a manner similar to that commonly used for Escherichia coli. Although the transformation efficiency for Agrobacterium is lower than that for E. coli, it is possible to obtain adequate numbers of Agrobacterium transformants with this technique. - [Read Transformation of Agrobacterium Using Electroporation Protocol]

Category: Bacteria Transformation Protocols

Protocol describes a method for transforming Agrobacterium with plasmid DNA using a freeze-thaw technique. Although the transformation efficiency for Agrobacterium is lower than that for Escherichia coli, it is possible to obtain adequate numbers of transformants with this technique. - [Read Transformation of Agrobacterium Using the Freeze-Thaw Method Protocol]

Category: Fungi Protocols: Neurospora Protocols

Simple method for growing Neurospora and for isolation of DNA that may be performed in two days from start to finish. The growth of mycelia in Petri plates eliminates the need for large numbers of flasks when growing many cultures for DNA isolation. - [Read Ultra-Fast Method of DNA Extraction from Neurospora Protocol]