isolated Protocols

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols: Tissue Preparation for LCM Protocols

LCM isolates specific cells or tissues from samples mounted on microscope slides. The samples are viewed through a thermoplastic film that is attached to a microcentrifuge tube lid. Localized heat, caused by the application of a laser pulse, fuses the membrane to the cells of interest, which can then be harvested for further analysis. RNA and proteins can be purified from the isolated cells, allowing detailed analysis of gene expression. This protocol is divided into three stages. - [Read (LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cel]

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

Simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and proteolytic treatment to permeabilize cells. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells for subsequent culturing based on DNA-content differences, is also described. Also presented are methods for staining of cell nuclei isolated from paraffin-embedded tissues, and deconvolution of DNA-content-frequency... - [Read Analysis of Cellular DNA Content by Flow Cytometry Protocol]

Category: Transfection Protocols: Stable Transfection Protocols

The AfCS is utilizing antisense technology to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. This can be achieved by the transfection of gene-specific antisense oligonucleotides (ASOs). The following procedure involves the transfection of ASOs into RAW 264.7 cells using FuGENE 6 transfection reagent. Subsequently, the isolated total RNA or protein from these transfected cells can be used to assess the level of mRNA or protein knockdown, respectively. - [Read Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish]

Category: PCR Protocols: Real-Time PCR Protocols

Protocol describes the real-time PCR using RNA purified from laser-captured tissues Laser Capture Microdissection (LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cells. - [Read cDNA Synthesis and Real-Time PCR Using RNA from Laser-Captured Cells Protocol]

Category: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes a method to collect early embryos from 6-week-old mice.  Subsequently, the isolated embryos can be injected with double-stranded RNA to induce knockdown of a gene of interest. - [Read Collection of Early Mouse Embryos for RNAi Protocol]

Category: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes a method to collect oocytes from 6-week-old mice.  Subsequently, the isolated oocytes can be injected with double-stranded RNA to induce knockdown of a gene of interest. - [Read Collection of Mouse Oocytes for RNAi Protocol]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Three Ambion kits were used to quantitate specific miRNAs and to detect differential miRNA expression in various mouse brain regions and cell types isolated by laser capture microdissection (LCM). These techniques can be applied to studying miRNA in other species, tissues, and cell types. Includes: Obtain Laser Capture Microdissected Samples; Isolate miRNA from LCM Samples; Quantitate miRNA by qRT-PCR. - [Read Detect and Quantitate MicroRNA in Laser Capture Microdissection Samples]

Category: Immunology

Protocol for Epstein-Barr Virus transformation of lymphoblasts. Protocol describes a method for the transformation of lymphoblasts by Epstein-Barr Virus (EBV). Cells may be isolated from whole blood or taken from cryopreserved, non-immortalized stocks. - [Read Epstein-Barr Virus Transformation of Lymphoblasts 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: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension. Hybridization technique which does not need formamide and dextran sulfate. As a model system, we used the repetitive specific human DNA probe pUC 1.77, labeled it with digoxigenin-11-dUTP by nick-translation, and hybridized it to metaphase chromosomes in suspension. These chromosomes were isolated by standard techniques from human lymphocytes. - [Read Fluorescence In Situ Hybridization of a Repetitive DNA Probe to Human Chromosomes in Suspension]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

This protocol describes a method for freezing ES cells in 96-well plates after ES colonies have been isolated by picking. - [Read Freezing Embryonic Stem (ES) Cells in 96-Well Plates Protocol]

Category: Immunology: Immunoprecipitation Protocols

Protocol for immunoprecipitation of mRNA-protein complexes. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. - [Read Immunoprecipitation of mRNA-Protein Complexes Protocol]

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: RNA Protocols: mRNA Extraction Protocols

Isolation of poly A+ RNA from Total RNA by Oligo(deoxythymidine)cellulose Chromatography. Total RNA is first isolated from the tissues or cells and then mRNA is isolated by PolyA+ selection using oligo(dT) cellulose. This is necessary for all tissue sources rich in RNase (and cell lines). Lazo Lab - [Read Isolation of poly A+ RNA from Total RNA by Oligo(deoxythymidine)cellulose Chromatography]

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]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

One step extraction for isolation of plant DNA. DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm2 in less than 20 min & no tube changes. Method was tested on several plant species. Method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated using standard DNA isolation. - [Read One-Step Isolation of Plant DNA Suitable for PCR Amplification]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Protein complexes can be isolated by several different approaches.  For example, a protein can be tagged with an epitope such as Flag or TAP and then overexpressed in a target cell, allowing the interacting proteins to be purified.  Similarly, epitope tags can be homologously recombined into the endogenous locus ("knocked-in"), allowing protein complexes containing the tagged proteins to be isolated at their natural expression level. - [Read Overview of Affinity Purification in Combination with Mass Spectrometry Protocol]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Peroxisome Isolation Protocols

To isolate peroxisomes from Saccharomyces cerevisiae of a quality sufficient for in vitro import studies, we optimized the conditions for cell growth and for cell fractionation. Stability of the isolated peroxisomes was monitored by catalase latency and sedimentability of marker enzymes. - [Read Peroxisomes in Saccharomyces cerevisiae]

Category: Plant Biology Protocols: Photosynthesis and Respiration Protocols

Protocol describing photosynthesis by respirometry. Includes characteristics of isolated chloroplasts. - [Read Photosynthesis by Respirometry]

Category: Viral Manipulation Protocols: Phage Protocols

Protocol describes a method for generating isolated plaques from a stock of bacteriophage lambda. Each plaque derives from infection of a single bacterium by a single bacteriophage particle. Because each plaque contains the progeny of a single virus particle, the bacteriophages derived from a single plaque are essentially genetically identical to one another. - [Read Plating Bacteriophage Lambda Protocol]

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: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

The double-stranded replicative form (RF) of bacteriophage M13 is isolated from infected cells using methods similar to those used to purify plasmid DNA. Several micrograms of RF DNA can be isolated from a 1-2-ml culture of infected cells. - [Read Preparation of Double-stranded (Replicative Form) Bacteriophage M13 DNA 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: 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]