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Long-Term Storage of Unfrozen Tetrahymena Cultures in Soybean Medium Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4472

Category: Tetrahymena Protozoa Protocols

Long-term storage of unfrozen Tetrahymena is convenient when a laboratory is not using the cells frequently; however, cells maintained under these conditions often take a couple of days of incubation in culture medium to recover. - [Read Long-Term Storage of Unfrozen Tetrahymena Cultures in Soybean Medium Protocol]

Long-Term Storage Storage of Unfrozen Tetrahymena Cultures in Maintenance Culture Medium Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4471

Category: Tetrahymena Protozoa Protocols

Protocol provides a method for long-term storage of Tetrahymena cultures using maintenance culture medium. - [Read Long-Term Storage Storage of Unfrozen Tetrahymena Cultures in Maintenance Culture Medium Protocol]

Lymphocyte Isolation and Culture Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4480

Category: Immunology: Mononuclear Cell Isolation Protocols: B Lymphocyte Isolation Protocols

Protocol describes a method for isolation and stimulation of lymphocytes. Leukocyte-rich plasma is collected from whole blood and then centrifuged through Ficoll-Hypaque. The collected cells are resuspended in growth medium containing various mitogens to stimulate growth. - [Read Lymphocyte Isolation and Culture Protocol]

Lysing Colonies and Binding of DNA to Filters Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3938

Category: Bacterial Protocols

Alkali is used to liberate DNA from bacterial colonies on nitrocellulose or nylon filters. The DNA is then fixed to the filter by UV-cross-linking or baking under vacuum. - [Read Lysing Colonies and Binding of DNA to Filters Protocol]

Lysing Tissue-Culture Cells for Immunoprecipitation Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4531

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]

Magnetic techniques for the isolation and purification of proteins and peptides - http://www.biomagres.com/content/2/1/7

Category: RNA Protocols: RNA-binding Protein Purification

Magnetic techniques for the isolation and purification of proteins and peptides. Safarik et al. 2004 - [Read Magnetic techniques for the isolation and purification of proteins and peptides]

Maintenance of Stocks of Marine Hypotrichs Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4469

Category: Laboratory Model Organisms Protocols

Protocol describes the preparation of stocks of marine hypotrichs for long-term storage. Marine euplotids are maintained by serial passage of tube stocks. - [Read Maintenance of Stocks of Marine Hypotrichs Protocol]

Making a Tetrad Dissection Needle Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4182

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

Microneedles attached to micromanipulators are used in the dissection of tetrads, isolation of zygotes from populations of mating haploid cells, and manipulation of individual cells. - [Read Making a Tetrad Dissection Needle Protocol]

Making Holding Pipettes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4394

Category: Microinjection Protocols: Microinjection Equipment

This protocol describes a method for making holding pipettes, to be used for microinjection. - [Read Making Holding Pipettes Protocol]

Making Holding Pipettes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4394

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol describes a method for making holding pipettes, to be used for microinjection. - [Read Making Holding Pipettes Protocol]

Making Injection Pipettes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4395

Category: Microinjection Protocols: Microinjection Equipment

This protocol describes the determination of useful settings with the Sutter Puller P-97 to make injection pipettes for microinjection. - [Read Making Injection Pipettes Protocol]

Making Injection Pipettes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4395

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol describes the determination of useful settings with the Sutter Puller P-97 to make injection pipettes for microinjection. - [Read Making Injection Pipettes Protocol]

Making Pipettes From Hard Glass Capillaries Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4354

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol describes a method for making pipettes from hard glass capillaries. - [Read Making Pipettes From Hard Glass Capillaries Protocol]

Mammalian Cell Culture and Preparation of Cells for RNAi in 24-Well Plates Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4341

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

This protocol introduces a method for maintaining mammalian cell culture suitable for treatment with siRNA. Transfection of cultured cells with siRNAs (see Transfection of siRNA Duplexes into Mammalian Cells) and downstream analysis of the knockdown cells. - [Read Mammalian Cell Culture and Preparation of Cells for RNAi in 24-Well Plates Protocol]

Mammalian Cell Culture and Preparation of Cells for RNAi in 24-Well Plates Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4341

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol introduces a method for maintaining mammalian cell culture suitable for treatment with siRNA. Transfection of cultured cells with siRNAs and downstream analysis of the knockdown cells. - [Read Mammalian Cell Culture and Preparation of Cells for RNAi in 24-Well Plates Protocol]

Mapping Dnase-I-hypersensitive Sites Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3949

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Protocol describes how isolated nuclei are incubated with varying amounts of Dnase I. Genomic DNA is then isolated from the nuclei and digested with a restriction enzyme, analyzed by gel electrophoresis, and probed by Southern hybridization. - [Read Mapping Dnase-I-hypersensitive Sites Protocol]

Mapping Protein-binding Sites on DNA by Dnase I Footprinting Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3947

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Dnase I is used to fragment a radiolabeled target DNA in the presence and absence of a nuclear extract. A "footprint" is generated when a protein binds to the target and protects a specific segment of DNA from the nucleolytic activity of Dnase I. By comparing the electrophoretic mobility of the Dnase I cleavage products to those of a sequence ladder derived from the same DNA fragment, the position(s) of the DNA sequences recognized by DNA-binding proteins can be determined. - [Read Mapping Protein-binding Sites on DNA by Dnase I Footprinting Protocol]

Mapping RNA with Nuclease S1 Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4053

Category: Nucleic Acid Purification Protocols

Preparations of RNA containing an mRNA of interest are hybridized to a complementary single-stranded DNA probe. At the end of the reaction nuclease S1 is used to degrade unhybridized regions of the probe, and the surviving DNA-RNA hybrids are then separated by gel electrophoresis and visualized by autoradiography or Southern hybridization. Method used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates. - [Read Mapping RNA with Nuclease S1 Protocol]

Mapping RNA with Ribonuclease and Radiolabeled RNA Probes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4054

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

Preparations of RNA containing an mRNA of interest are hybridized to a radiolabeled single-stranded RNA probe. The method can be used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates. - [Read Mapping RNA with Ribonuclease and Radiolabeled RNA Probes Protocol]

Markers for Pulsed-field Gel Electrophoresis Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3222

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol fopr markers of pulsed-field gel electrophoresis. Markers for pulsed-field gel electrophorsis can be generated by ligation of linear monomers of bacteriophage {lambda} DNA (48.5 kb) into a nested series of concatemers. This procedure yields a series of concatemers that contain up to 20 tandemly arranged copies of bacteriophage DNA. - [Read Markers for Pulsed-field Gel Electrophoresis Protocol]

Mass Culture of Freshwater Hypotrichs Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4466

Category: Laboratory Model Organisms Protocols

Protocol describes the culture of freshwater hypotrichs using the alga Chlorogonium elongatum as a food source. - [Read Mass Culture of Freshwater Hypotrichs Protocol]

Measurement of CAT in Extracts of Mammalian Cells Using Thin-layer Chromatography - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3951

Category: Reporter Gene Assays: Cat Assays Protocols

In this protocol, extracts prepared from cells transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid are incubated with radiolabeled chloramphenicol. The acetylated products generated by the action of CAT are separated from the unmodified drug by thin-layer chromatography and quantitated by scraping the spots from the thin-layer plates and counting them by scintillation spectroscopy. - [Read Measurement of CAT in Extracts of Mammalian Cells Using Thin-layer Chromatography]

Measurement of DNA Content Using Propidium Iodide (PI) Staining of Fixed Whole Cells Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4436

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol describes a method for quantitative measurement of DNA using propidium iodide (PI) staining and flow cytometry. PI stains all double-stranded regions of both DNA and RNA by intercalating between the stacked bases of the double helix. PI cannot penetrate an intact cell membrane; therefore, cells are fixed prior to staining. The ethanol-fixed cells can be stored unstained at 4°C for days, or even weeks, and then stained and analyzed. - [Read Measurement of DNA Content Using Propidium Iodide (PI) Staining of Fixed Whole Cells Protocol]

Measurement of GFP Expression and DNA Content in Permeabilized Cells - http://cyto.mednet.ucla.edu/Protocols/DNA%20and%20GFP%20PI.htm

Category: Reporter Gene Assays: GFP Assay Protocols

Protocol measuring the amount of GFP expression and DNA content in permeabilized cells. Includes: Fix cells with formaldehyde; Permeabilize cells with ethanol; Preparation of Buffered 2% Formaldehyde Solution. - [Read Measurement of GFP Expression and DNA Content in Permeabilized Cells]

Measurement of GFP Expression and DNA Content in Permeabilized Cells Protocol - http://cyto.mednet.ucla.edu/Protocols/DNA%20and%20GFP%20PI.htm

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

Protocol for the measurement of GFP expression and DNA content in permeabilized cells. Includes: Fix cells with formaldehyde; Permeabilize cells with ethanol; Preparation of Buffered 2% Formaldehyde Solution. - [Read Measurement of GFP Expression and DNA Content in Permeabilized Cells Protocol]

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In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.

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