preparations Protocols

Category: Immunology: B Cell Protocols

Procedure permits the isolation of at least 5 µg of total RNA from a sample of purified mouse splenic B lymphocytes. The quality of the RNA is assessed by separation of an aliquot through 1% agarose and staining with ethidium bromide as described in AfCS protocol Visualization of RNA Preparations on 1% Agarose Gels. The isolated RNA is used for analysis of gene expression by microarray technology. analysis of gene expression by microarray technology. - [Read Preparation of B-Lymphocyte RNA for Microarray Analysis Protocol]

Category: Cytogenetics Protocols

An ideal method of tissue preparation ensures both good specimen morphology and that the target molecules are in the optimum state for probe access and hybridization. DNA:DNA in situ hybridization is usually carried out on chromosome spread preparations where chromosome and nuclei are released from cells and spread on a glass microscope slide. This method yields well separated and enlarged chromosomes with good morphology which can be analyzed in transmitted light or fluorescence microscopes. - [Read Preparation of Chromosome Spreads]

Category: Plant Biology Protocols: Arabidopsis Tissue Culture

Protocol for the evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls. - [Read Procedure for Purifying Cell Walls from Arabidopsis Hypocotyls]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Immunoaffinity purification of antibodies is used to purify antigen-specific antibodies from a preparation of polyclonal antibodies.  Such purification is commonly needed in the production of antipeptide antibodies, where it is used to concentrate the desired antibodies and separate them from those raised against carrier proteins.  It is also used for the more general purpose of removing unwanted, nonspecific binding activity from polyclonal antibody preparations. - [Read Purification of Antibodies on an Antigen Column Protocol]

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

Crude preparations of plasmid DNA are first treated with lithium chloride and Rnase (to remove RNA).  The plasmid DNA is then precipitated in a solution containing polyethylene glycol and MgCl2. - [Read Purification of Plasmid DNA by Precipitation with Polyethylene Glycol Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Protocol for the quality control of chromosome preparations. - [Read Quality Control of Chromosome Preparations Protocol]

Category: Nucleic Acid Purification Protocols

In this protocol, ultrafiltration through Centricon or Microcon concentrators is used to remove unused primers, primer-dimers, and NTPs from preparations of amplified DNA. - [Read Removal of Oligonucleotides and Excess dNTPs from Amplified DNA by Ultrafiltration Protocol]

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

Contamination of plasmid DNA by fragments of DNA and RNA is reduced to an acceptable level by centrifugation through 1 M sodium chloride.  This method was devised by Brian Seed when he was a graduate student at Harvard University. - [Read Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Centrifugation]

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

High-molecular-weight RNA and proteins can be precipitated from preparations of plasmid DNA by high concentrations of LiCl and removed by low-speed centrifugation. - [Read Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Li]

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

This method reproducibly yields several micrograms of yeast DNA that can be efficiently cleaved by restriction enzymes and used as a template in PCR. - [Read Small-scale Preparations of Yeast DNA Protocol]

Category: Neuroscience Protocols: Histology Stains Protocols: Giemsa Stains Protocols

Protocol stain for air dried cytology preparations. - [Read Stain for Air Dried Cytology Preparations Protocol]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for the storage of chromosome preparations for FISH. Includes: Cell Pellet; Chromosome Slide Preparation. - [Read Storage of Chromosome Preparations for FISH Protocol]

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

Protocol describes methods for maintaining healthy, dividing mammalian cells in culture and during imaging, when mitosis can be examined. Rose chambers are preferable for observation and microinjection of living mitotic cells, but slide/coverslip preparations are easy to make and do not require any special equipment. - [Read Studying Mitosis in Cultured Mammalian Cells Protocol]

Category: Cytogenetics Protocols

Protocol describes methods for maintaining healthy, dividing mammalian cells in culture and during imaging, when mitosis can be examined. Rose chambers are preferable for observation and microinjection of living mitotic cells, but slide/coverslip preparations are easy to make and do not require any special equipment. Another inexpensive and easy-to-use alternative is to grow cells in a culture dish with a glass bottom. Such dishes are suitable for microinjection experiments. - [Read Studying Mitosis in Cultured Mammalian Cells Protocol]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: Mitosis Protocols

Protocol describes methods for maintaining healthy, dividing mammalian cells in culture and during imaging, when mitosis can be examined. Rose chambers are preferable for observation and microinjection of living mitotic cells, but slide/coverslip preparations are easy to make and do not require any special equipment. Another inexpensive and easy-to-use alternative is to grow cells in a culture dish with a glass bottom. Such dishes are suitable for microinjection experiments. - [Read Studying Mitosis in Cultured Mammalian Cells Prtocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocol describes removal of T cell subsets by cytotoxic elimination using CD-specific antibodies. Describes the complete removal of T cells from lymphocyte preparations based on the presence of the glycoprotein Thy-1 on the cell surface of T lymphocytes. Cytotoxic elimination is employed; however, Thy-1-specific antibodies are used rather than MHC class II-specific antibodies so that T cells are eliminated rather than B cells and accessory cells. - [Read T Cell Depletion by Cytotoxic Elimination Protocol]

Category: Immunohistochemistry Protocols: Antigen Retrieval Protocols

Fixation can mask epitopes. However, it is often possible to re-expose them using a gentle incubation with proteases, which removes obstructing structures and allows antibody access, as described here. Many proteases can be used for this procedure, including very crude preparations of proteases, such as pronase. However, using a better-characterized protease, such as trypsin, allows a more controlled reaction and better comparison between experiments. - [Read Unmasking Hidden Epitopes with Proteases Protocol]

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Method is used to assess (roughly) the integrity of total RNA samples by visualization of discreet 18S and 28S ribosomal RNAs.  Total RNA is separated by electrophoresis through a 1% agarose gel containing 1.3 ìM ethidium bromide.  Binding of the ethidium bromide to the RNA allows visualization of the separated RNA molecules when the gel is exposed to ultraviolet (UV) light. - [Read Visualization of RNA Preparations on 1% Agarose Gels Protocol]

Category: Neuroscience Protocols: Histology Stains Protocols

Protocol stain for wet fixed cytology preparations: Papanicolaou Method. - [Read Wet Fixed Cytology Preparations Protocol]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes a method for staining nerve fibers in whole-mount preparations of avian embryos using an antibody against the 160-kD subunit of neurofilaments.  This allows the comparison of the branching pattern of motor and sensory neurons between control and experimental embryos.  This protocol has been successfully applied for embryos at different stages up to about stage 33 (7 days of incubation). - [Read Whole-Mount Preparations for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos]

Category: Avian Bird Protocols

Protocol describes a method for staining nerve fibers in whole-mount preparations of avian embryos using an antibody against the 160-kD subunit of neurofilaments. This allows the comparison of the branching pattern of motor and sensory neurons between control and experimental embryos. This protocol has been successfully applied for embryos at different stages up to about stage 33 (7 days of incubation). - [Read Whole-Mount Preparations for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos Protocol]