preparations Protocols

Category: Buffers Media, and Solutions

A-Z Chemical Preparations.Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College. - [Read A-Z Chemical Preparations.]

Category: Signalling Signal Transduction Protocols

Technique for Alkaline Phosphatase (APAAP). Techniques includes information on: Cytological Preparations, Fixation, Technique, Results, Substrate Solution and 0.005M Tris/HC1 Saline Buffer pH 7.6. - [Read Alkaline Phosphatase (APAAP) Technique]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Method is for preparing chromosomes from single flower buds of A. thaliana. It does not kill the plants allowing the determination of their chromosome number throughout development. Includes: Preparations of Arabidopsis; Preparation of Chromosome; Staining Chromosomes. - [Read Chromosome Spreads from Flower Buds of Arabidopsis thaliana Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Protocol for combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations. Includes: Cell preparations and BrdU labeling; Detection of antigen by immunocytochemistry (ICC); Visualization of ICC antigen; -Gal-BCIG reaction (for producing a blue precipitate visible under brightfield microscopy); Cell processing for in situ hybridization; In situ hybridization (ISH); etc... - [Read Combined DNA In Situ Hybridization and Immunocytochemistry Protocol]

Category: Immunology: Immunoprecipitation Protocols: Immunoprecipitation of Radiolabeled Cells Protocols

Most powerful and convincing method to determine if a specific protein is phosphorylated in a physiologically relevant manner is to assay phosphorylation in situ. The procedure described involves incubating cultured cells (e.g., primary neuronal cultures or transfected cells) or tissue preparations (e.g., hippocampal slices) with [32P]orthophosphate, which is then taken up by the cells or tissues and incorporated into the γ-phosphate position of ATP. - [Read Detection of Protein Phosphorylation in Tissues and Cells Protocol]

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Protocol uses northern blot technology to detect short interfering RNAs (siRNAs) or micro-RNAs (miRNAs) in RNA preparations from Caenorhabditis elegans. - [Read Detection of si/miRNA in C. elegans by Northern Blot Protocol]

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Protocol uses Rnase protection to detect short interfering RNAs (siRNAs) in RNA preparations from Caenorhabditis elegans.  SiRNAs can also be detected by northern blot.  However, the Rnase protection assay seems to be more sensitive. - [Read Detection of siRNA in C. elegans Using Rnase Protection Protocol]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for dot and slot hybridization of purified RNA. Dot blotting of RNA is best carried out using purified preparations of RNA that are denatured with glyoxal or formaldehyde immediately before loading onto a nylon membrane through a vacuum manifold. - [Read Dot and Slot Hybridization of Purified RNA Protocol]

Category: Microscopy Protocols: Electron Microscopy Protocols

Article describe the preparation of cells for correlative electron microscopy after live light microscopic observation of fluorescently labeled cytoskeletal proteins microinjected into the same cells. Since identification of cytoskeletal elements in electron microscopic preparations is an essential part of any correlative study, procedures for immunogold labeling of cytoskeletal components and for myosin S1 decoration of actin filaments are also described. - [Read Electron Microscopy of the Cytoskeleton of Cultured Cells]

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

Protocol describes how monocytes can be sorted from a lysed whole blood sample, and that cytospin preparations can be made for examination by light microscopy. - [Read Enrichment Of Monocytes For Morphological Study Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Formamide can be used instead of proteinase K to dissociate bacteriophage {lambda} coat proteins from the viral {lambda}. The procedure is rapid and works best with large-scale preparations of bacteriophage {lambda}. - [Read Extraction of Bacteriophage lambda DNA from Large-scale Cultures Using Formamide Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

DNA is best isolated from bacteriophage lambda particles by digesting the viral coat proteins with a powerful protease such as proteinase K, followed by extraction with phenol:chloroform. This procedure is easily adapted for use with smaller-scale preparations of bacteriophage lambda. - [Read Extraction of Bacteriophage lambda DNA from Large-scale Cultures Using Proteinase K and SDS Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Most manipulations with M13, including preparations of viral stocks and isolation of single- and double-stranded DNAs, begin with small-scale liquid cultures that are infected with an M13 plaque, picked from an agar plate. - [Read Growing Bacteriophage M13 in Liquid Culture Protocol]

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Homogeneous caspase-3/7 assay. Includes: Detection of Caspase-3/7 Activity in Cell Culture;  Detection of Caspase-3 or -7 Activity in Purified Caspase Preparations; Positive and Negative Cell Culture Controls. - [Read Homogeneous Caspase 3-7 Assay Protocol]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to do film and digital imaging of microscopic preparations. - [Read How to Do Film and Digital Imaging of Microscopic Preparations]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

The basis of this procedure is that two specific cell type preparations may be isolated, exposed separately to various compounds over a range of concentrations, and the cytotoxicity of these determined.  Parameters deemed indicative of a cytotoxic effect include a reduction in de novo protein synthesis and decreased glucose and fatty acid metabolism.  A cytotoxic effect may indicate that a chemical is likely to be nephrotoxic in vivo. - [Read Isolated Rat Glomeruli and Proximal Tubules]

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: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Leukostat Staining of Cytospin Preparations to Detect Apoptosis. Shailaja Kasibhatla et al. Leukostat staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a light microscope and counted to quantify apoptosis. This protocol can be used both for cells that grow in suspension and for adherent cells. - [Read Leukostat Staining of Cytospin Preparations to Detect Apoptosis]

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

This protocol has been established to maintain the efficient use of the AutoMACS cell sorter during periods of heavy use (e.g., three preparations of splenocytes from 16 spleens per week). Includes: Running the SAFE Clean Program; Clearing Debris. - [Read Maintenance of the AutoMACS Cell Sorter Protocol]

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]

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]

Category: Cytogenetics Protocols

Protocol for meiotic preparations from Testis. - [Read Meiotic Preparations from Testis Protocol]

Category: Cytogenetics Protocols

Protocol for mitotic chromosome preparations from bone marrow. - [Read Mitotic Chromosome Preparations from Bone Marrow Protocol]

Category: Cytogenetics Protocols

Protocol for mitotic chromosome preparations from mammalian suspension cell cultures. - [Read Mitotic Chromosome Preparations from Mammalian Suspension Cell Cultures Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Protocol for multiple-target DNA in situ hybridization with enzyme-based cytochemical detection systems. Includes: Cell preparations; Cell processing; Probe preparation; Multiple-target in situ hybridization (ISH); ISH with separate probe and target denaturation [for probes with repetitive (e.g., Alu) elements]; Post-hybridization washes; Enzyme-based cytochemical detection; etc.. - [Read Multiple-Target DNA In Situ Hybridization with Enzyme-Based Cytochemical Detection Systems Protocol]