grown Protocols

Category: Plant Biology Protocols: Plant Genetics Protocols

For analysis of metaphase chromosomes, any tissue containing dividing cells can be used: Root tips from young seedlings, from newly grown roots at the edge of plant pots or hydroponic culture are all suitable. Alternatively, flower buds, anthers, carpels or leaf or apical meristems can be used. Includes metaphase arresting reagents. - [Read Accumulation and Fixation of Plant Metaphase Chromosomes Protocol]

Category: DNA Protocols: Cosmid Library Protocols

Amplification of cosmid libraries may result in distorted representation of cloned genomic sequences and should be avoided wherever possible. In this method of amplification, distortion of the library is rarely a problem because at no stage are bacteria containing different recombinant cosmids grown in competition with one another. - [Read Amplification and Storage of a Cosmid Library: Amplification on Filters Protocol]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

Method describes how a crude extract is prepared, and the activity is normalized to the amount of protein assayed. This method is particularly suitable for comparing cells that are grown under very different conditions or that have different genetic backgrounds. - [Read Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts]

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

Protocol describes how a crude extract is prepared, and the activity is normalized to the amount of protein assayed. This method is particularly suitable for comparing cells that are grown under very different conditions or that have different genetic backgrounds. - [Read Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts Protocol]

Category: Yeast Protocols: Yeast Protein Protocols

This assay is performed to detect ubiquitylated proteins in yeast. Yeast that have been transformed with a vector expressing polyhistidine-tagged ubiquitin (Ub) under the control of a copper-inducible promoter are grown, induced with copper, and harvested. Total ubiquitylated proteins are then recovered by nickel-affinity chromatography, and specific proteins are detected by Western blotting. - [Read Detection of Ubiquitylated Proteins in Yeast Protocol]

Category: Cell Culture Protocols

For low-resolution work, cells to be used for staining can be grown directly on regular tissue-culture dishes. It is a convenient method that does not require much preparatory work. - [Read Growing Adherent Cells Directly on Tissue Dishes Protocol]

Category: Laboratory Model Organisms Protocols

Protocol describes the growth and concentration of the alga Chlorogonium elongatum as a food source for culturing freshwater hypotrichs. Most freshwater hypotrichs (including Oxytricha nova, O. fallax, and O. trifallax; Euplotes aediculatus and E. eurystomous; and Stylonychia lemnae) can be grown to high density with Chlorogonium as the food organism. A similar regimen can be used to prepare other food sources such as Tetrahymena or bacteria (e.g., Aerobacter aerogenes). - [Read Growth and Concentration of Chlorogonium for Culturing Freshwater Hypotrichs Protocol]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

This protocol describes a method for isolating individual ES cell colonies by picking after they have grown on selective medium. - [Read Isolating Individual Embryonic Stem (ES) Cell Colonies by Picking Protocol]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: MEF Protocols Mouse Embryonic Fibroblasts

Embryonic stem cells are usually grown on a layer of mitotically inactivated primary mouse embryonic fibroblasts to promote growth and prevent differentiation. Boris Greber - [Read Isolation and handling of primary mouse embryonic fibroblasts (MEFs)]

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

Protocol for isolation of DNA from mammalian cells grown in 96-well microtiter plates. Each well yields sufficient genomic DNA for several standard PCRs or for analysis in a single lane of a Southern hybridization. - [Read Isolation of DNA from Mammalian Cells Grown in 96-well Microtiter Plates Protocol]

Category: C. elegans Protocols: C. elegans Cell Culture Protocols

Protocol for liquid culture of worms. Includes: superbroth; S- basal; worm plates; GROWING THE BACTERIA (WORM FOOD); GROWING THE WORMS; AFTER THE CULTURE HAS GROWN; PREPARING EGGS TO START SYNCHRONIZED LIQUID CULTURES. - [Read Liquid Culture of Worms Protocol]

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

Yeast strains may be stored indefinitely at low temperatures (-80 degrees C). Two archiving methods are presented below. In Method A , the cells are grown on a plate, while in Method B the cells are grown in liquid culture. - [Read Long Term Storage of Yeast Stocks Protocol]

Category: Cell Culture Protocols: Cell Lysis Protocols

For cells grown in tissue culture, the most useful method of lysis is treating with detergents, as described in this protocol. Non-ionic detergents, such as NP-40, solubilize the plasma and intracellular membranes, break many weak intermolecular bonds, and solubilize most of the commonly studied protein antigens. RIPA lysis buffer may be used as a more rigorous extraction buffer to release all but the insoluble proteins of the cell and to break most weak noncovalent interactions. - [Read Lysing Tissue-Culture Cells for Immunoprecipitation Protocol]

Category: Fungi Protocols: Fungi Genetics Protocols

Quick and reliable method to analyze meiotic segregation patterns in Coprinus cinereus using the polymerase chain reaction. The advantages of this method include: 1. The tissue is grown and lyophilized in the same tube, which facilitates the simultaneous analysis of many segregants. 2. Only one extraction step is necessary. 3. The markers are scored by gel electrophoresis, thereby bypassing Southern analysis. - [Read Method to Analyze Meiotic Segregation Patterns in Coprinus cinereus Using PCR]

Category: Cell Culture Protocols: Cell Lines Protocols

RAW 264.7 cells are a macrophage-like, Abelson leukemia virus transformed cell line derived from BALB/c mice. For routine maintenance in culture (passage), cells are seeded at a confluence of approximately 10% (1 x 106 and 3 x 106 cells in 100-mm and 150-mm plates, respectively) and grown to a confluence of approximately 80%. This procedure requires the cells to be split every two days. - [Read Passage Procedure for RAW 264.7 Cells]

Category: Bacteria Transformation Protocols

Protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to 3 x 108 transformed colonies/µg of plasmid DNA. The protocol works optimally when the bacterial culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can be set up in a 4°C cold room and regulated to 18°C. - [Read Preparation and Transformation of Competent E. Coli: "Ultra-Competent" Cells Protocol]

Category: E Coli Protocols: E coli Transformation Protocols

Protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to 3 x 108 transformed colonies/µg of plasmid DNA. The protocol works optimally when the bacterial culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can be set up in a 4°C cold room and regulated to 18°C. - [Read Preparation and Transformation of Competent E. Coli: "Ultra-Competent" Cells Protocol]

Category: Cell Biology and Cell Culture: Cell Culture Techniques: Cell Line Preservation Freezing

Cell lines are reactivated and grown to a count of 1 x 108 cells. The cells are pelleted and stored frozen at -80 degrees C prior to DNA extraction. Donis-Keller. - [Read Reactivating Cell Lines and Cell Growth for DNA Preparation]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol describes a split luciferase complementation assay that can be used to repetitively and noninvasively study the interaction of proteins in small living animals. After the expression of the appropriate vectors has been checked in cell culture in vivo, studies can be performed either by implanting transiently transfected cells for short-term analysis (maximum of 7 days), or with tumor models grown from tumor cells stably expressing the complete reporter system. - [Read Split Luciferase Complementation Assay for Studying Interaction of Proteins X and Y in Living Mice]

Category: Cell Culture Protocols: Trypsinization Protocols

Protocol describes the trypsinization of cells in monolayer culture to facilitate subculture or harvesting. To avoid cross-contamination of cells, it is important for each cell line to be subcultured independently. No more than one cell line should be in the tissue culture hood at any one time. - [Read Trypsinization of Cells Grown in Monolayer Protocol]