content Protocols

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: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast DNA Isolation Protocols

Protocol describes the preparation of genomic DNA from a 5-ml liquid culture of yeast cells. - [Read Preparation of Genomic DNA from Yeast Using Glass Beads Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

In this protocol, a bacterial lysogen is constructed from a recombinant bacteriophage {lambda} encoding a fusion protein of interest. The resulting lysogenic colonies are induced to synthesize the fusion protein, which is then isolated in preparation for functional and biochemical analyses. - [Read Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage {lambda} Lysogens]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

This rapid method is used to screen bacteriophage {lambda}gt11 clones for the production of immunodetectable fusion proteins. After optimizng the conditions of infection and induction, the method can be used to produce preparative amounts of a fusion protein. - [Read Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage {lambda}: Lytic Infection]

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

MEF feeders are prepared weekly to provide a substrate for undifferentiated embryonic stem (ES) cells. Primary MEF cells are thawed, established in culture, treated with mitomycin C to halt their proliferation so they cannot overgrow the ES cultures, and then replated onto dishes convenient for ES cell culture. This protocol can also be used to prepare feeder cells from STO fibroblast cell lines. - [Read Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates Protocol]

Category: Signalling Signal Transduction Protocols

Protocol provides a method for acheiving a sufficient sample for several determinations of cAMP. The protocol described for measuring the content of cyclic adenosine 3',5'- monophosphate (cyclic AMP or cAMP) in cardiac myocytes is an enzyme-linked immunoassay system. Protocol includes information on: Treatment of Cells and Preparation of Extracts; Use of Environmental Chamber; Reagents and Materials. - [Read Preparation of Myocyte Lysates for Cyclic AMP Determination]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with alkali and SDS. - [Read Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is isolated from intermediate-scale (20-50 ml) bacterial cultures by treatment with alkali and SDS. - [Read Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with alkali and SDS. - [Read Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating.  This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains). - [Read Preparation of Plasmid DNA by Large-scale Boiling Lysis Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Large (>15 kb), closed circular plasmids are prepared (albeit inefficiently and in small yield) by lysing bacteria with SDS. - [Read Preparation of Plasmid DNA by Lysis with SDS Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating.  This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains). - [Read Preparation of Plasmid DNA by Small-scale Boiling Lysis Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar media with toothpicks. - [Read Preparation of Plasmid DNA: Toothpick Minipreparation Protocol]

Category: Antibody Protocols: Antibody Production Protocols

Polyclonal antibodies can be isolated from animal plasma or serum using the procedure described in this protocol. The Gradiflow BF400 instrument has two liquid streams that circulate through a separation cartridge positioned between two electrodes and composed of three hydrogel polyacrylamide membranes, which define the channels for the two sample streams. The central membrane forms a physical barrier between the two streams. - [Read Preparation of Polyclonal Antibodies from Plasma or Serum Using the Gradiflow BF400]

Category: Signalling Signal Transduction Protocols

The protocol provides a method to achieve a sample sufficient for one determination of cAMP using the acetylation protocol. The protocol describes the method used for measuring the content of cyclic adenosine 3',5'- monophosphate (cyclic AMP or cAMP) in RAW 264.7 cells using an enzyme-linked immunoassay system. Information included in the protocol: Treatment of Cells and Preparation of Extracts; Reagents and Materials. - [Read Preparation of RAW 264.7 Lysates for Cyclic AMP Determination]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

The method uses Trizol, a phenol-base reagent produced by Invitrogen. - [Read Preparation of RNA from Paraffin-Embedded Fixed Tissue Using Trizol Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

Tissues are homogenized in guanidinium isothiocyanate lysis buffer and then subjected to cesium chloride ultracentrifugation. The resulting RNA is then extracted with phenol and chloroform to remove protein and lipid contaminants. - [Read Preparation of RNA from Plant Tissue Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifug]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

Protocol is based on the standard Trizol protocol for the purification of RNA from animal cells using Trizol (Purification of RNA from Animal Cells using Trizol). In this version, adapted for use with plant tissues, a high-salt isopropanol precipitation step has been added to precipitate RNA selectively, while maintaining polysaccharides and proteoglycans in solution. - [Read Preparation of RNA from Plant Tissue Using Trizol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected cells into the surrounding medium. The filamentous particles are concentrated by precipitation from a high-ionic-strength buffer with polyethylene glycol. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. This protocol is generally used to prepare single-stranded DNA from a small number of M13 isolates. - [Read Preparation of Single-stranded Bacteriophage M13 DNA Protocol]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

siRNAs produced upon the addition of dsRNA to Drosophila embryo extract are enriched in a micrococcus-nuclease-resistant fraction.  After proteinase K treatment and dephosphorylation with calf intestinal phosphatase, these siRNAs mediate efficient RNAi in vitro. - [Read Preparation of siRNAs from Drosophila Embryo Extracts Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Single-stranded templates of bacteriophage M13 DNA containing 20-30 residues of uracil in place of thymine are generated during growth of the bacteriophage in an F' strain of E. coli carrying mutations in the ung and dut genes. This DNA is used as a template in the Kunkel method of oligonucleotide-directed mutagenesis (Oligonucleotide-directed Mutagenesis of Single-stranded DNA). - [Read Preparation of Uracil-containing Single-stranded Bacteriophage M13 DNA Protocol]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols

Flow cytometry is used to analyze the quantity of DNA in cells. Since the DNA content of cells varies through the cell cycle, this information can provide an indication of cell cycle progression. This protocol uses SYTOX Green staining. - [Read Preparation of Yeast Cells for Flow Cytometry Protocol]

Category: Plant Biology Protocols: Arabidopsis Transformation Protocols

Protocol for an improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. - [Read Preparing Agrobacterium Cells That Simplifies the Arabidopsis Transformation Protocol]

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

Staining of tissues can be carried out on cell smears prepared from biopsy samples, such as needle aspirates, tissue scrapings, or freshly dissected tissues. - [Read Preparing Cell Smears from Tissue Samples for Immunostaining Protocol]

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

Protocol describes how double-stranded plasmid DNA is denatured by alkali and annealed to an appropriate oligonucleotide primer, in preparation for dideoxy-sequencing reactions catalyzed by Sequenase. - [Read Preparing Denatured Templates for Sequencing by Dideoxy-mediated Chain Termination Protocol]