isolation Protocols

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

The first part of the isolation procedure is a flotation through a continuous iodixanol gradient; this gradient is essentially a resolving gradient in which the caveolin-rich vesicles are concentrated in the top third of the gradient, while the predominantly caveolin-poor vesicles band in denser regions. A second discontinuous gradient is essentially a concentration gradient to band the caveolin-rich vesicles sharply at an interface. - [Read Purification of Caveolae Membranes from a Plasma Membrane Fraction of Cultured Cells and Tissues]

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

This protocol uses a "light mitochondrial" pellet from a mammalian liver homogenate. The gradient thus has to resolve a variety of denser components (peroxisomes, lysosomes, mitochondria) from the Golgi membranes, which have a low density in iodixanol (1.06-1.09 g/ml) [1]. The protocol is specifically tailored to the purification of Golgi membranes from this pellet and is unsuitable for the isolation or analysis of other organelles present in the light mitochondrial fraction. - [Read Purification of Golgi Membranes from a Light Mitochondrial Fraction in a Self-Generated Gradient]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

Although Percoll gradients were able to provide a purified sporocyst fraction, because these particles do not all band in a discrete manner in such gradients, they were unable to provide a simultaneous isolation of a pure oocyst wall fraction. Gradients formed from this protocol on the other hand are able to provide purified sporocysts and oocyst walls in the same gradient. - [Read Purification of Oocyst Walls and Sporocysts from Toxoplasma gondii Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

This protocol is not phenol-based, but does require the addition of chloroform. The Concert Plant Reagent is intended for the isolation of RNA from a wide variety of plant tissues including blue spruce needles, potato tuber etc. - [Read Purification of RNA from Plant Tissue Using the Concert Plant Reagent]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Mammalian DNA prepared from blood or tissues as described in this protocol is 20-50 kb in size and suitable for use as a template in PCRs.  The yields of DNA vary between 0.5 and 3.0 µg/mg tissue or 5 and 15 µg per 300 µl of whole blood. - [Read Rapid Isolation of Mammalian DNA Protocol]

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

This method is used to isolate genomic yeast DNA or shuttle plasmids that replicate in both S. cerevisiae and E. coli. The DNA can be used as a template for PCR and for transformation. - [Read Rapid Isolation of Yeast DNA Protocol]

Category: Mouse Protocols: Mouse Cell Culture Protocols

Protocol for rat Chromaffin cells primary cultures: Standardization and quality assessment for single-cell assays. Includes: Preliminars; Isolation of the rat adrenal medulla; Enzymatic digestion of medullary tissue; Collecting and culturing the cells. - [Read Rat Chromaffin Cells Primary Cultures Protocol]

Category: Neuroscience Protocols: Neuronal Cell Culture Protocols: Rat Neuronal Cell Culture Protocols

Protocol for standardization of both the isolation procedure and the quality of the cultures. - [Read Rat Chromaffin Cells Primary Cultures Standardization and Quality Assessment for Single Cell Assays]

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

Protocol to be used as reference to help standardization of both the isolation procedure and the quality of the cultures allowing a better comparison of the results obtained from different laboratories. - [Read Rat Chromaffin Cells Primary Cultures: Standardization and Quality Assessment for Single-Cell Assay]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol for RNA affinity isolation with PUMILIO from Drosophila melanogaster. - [Read RNA Affinity Isolations with PUMILIO from Drosophila melanogaster Protocol]

Category: Immunology: Immunoprecipitation Protocols

Protocol for RNA co-immunopurification with specific RNA-binding proteins. Includes: Binding to IgG beads; Washing the beads; Elution with TEV-protease; RNA isolation. - [Read RNA co-immunopurification with specific RNA-binding proteins]

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

Protocol describes the use of RT-PCR to confirm knockdown of a gene of interest in RNAi-treated mouse oocytes and early embryos.  All of the solutions used during RNA isolation and reverse transcription must be Rnase-free - [Read RNA Isolation and RT-PCR from dsRNA-Treated Mouse Oocytes and Early Embryos Protocol]

Category: DNA Protocols: cDNA Library Protocols

Protocol for RNA isolation for cDNA library construction. - [Read RNA Isolation for cDNA Library Construction Protocol]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols

Protocol for RNA isolation. - [Read RNA Isolation Protocol]

Category: Cell Biology and Cell Culture

Protocol for the isolation of the lipid-rich microdomains of the plasma membrane, notably caveolae and lipid rafts. Methods for the isolation of lipid rafts are based on the insolubility of these structures in the nonionic detergent TritonX-100. Either the intact cells are treated with a detergent-containing solution or a post-nuclear supernatant is prepared from a cell homogenate and then Triton X-100 is added to this supernatant. - [Read S20 Purification of detergent-insoluble lipid rafts from cells and tissues.]

Category: Fungi Protocols: Aspergillus Protocols

Protocol allows the isolation and enumerate Aspergillus spp spores in air. Includes: Sampling Procedure; Sampling Location; Selection of Sampling Time; Sampling Steps; Laboratory Procedure; Enumerating the Colony Forming Units. - [Read Sampling of Aspergillus spp Spores in Air Protocol]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Germ layers are multipotent tissues that have the ability to differentiate into various tissue types. Isolation and manipulation of germ layers is important for examining differentiation. This protocol describes a method for separating postimplantation germ layers. - [Read Separating Postimplantation Germ Layers Protocol]

Category: Mouse Protocols: Mouse Genetics Protocols

Protocol describes a simple procedure for DNA fragment isolation and purification. The resulting DNA fragment can then. be used for microinjection to produce transgenic mice - [Read Simple, Reliable Steps for DNA Fragment Isolation and Purification Protocol]

Category: Fungi Protocols: Neurospora Protocols

DNA isolation method yields an average of 0.6 micrograms of genomic DNA that is suitable for Southern analysis or PCR. Starting with fresh mycelium, 20 to 40 samples can be processed in approximately two hours. Better yields (about 5 micrograms) may be obtained by suspending approximately 100 microliters of ground lyophilized mycelium in 500 microliters of isolation buffer and following the protocol. - [Read Small Scale DNA Preps for Neurospora crassa Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

Protocol describing isolation of small scale plasmid from Xanthomonas. - [Read Small Scale Plasmid Isolation for Xanthomonas]

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

Protocol for spore germination. This procedure is typically used for the isolation and preparation of spores from a diploid strain heterozygous for a marked disruption (e.g., yfg1::his3+) Inoculation of the spore population into minimal medium lacking the nutritional supplement corresponding to the disruption marker (e.g., minimal medium lacking histidine) allows only the disruption spores to germinate. - [Read Spore Germination Protocol]

Category: Primary Cell Isolation and Culture Protocols

The protocol is one example of differential gene expression analysis of cells obtained from microdissected tissue. Includes: Microdissection and RNA Isolation; Reverse Transcription; PCR; P.A.G.E.; Sequencing of Differentially Expressed Bands. - [Read Targeted Differential Display Protocol]

Category: Primary Cell Isolation and Culture Protocols

This method enables the isolation of different populations of liver cells and describes their subsequent culture. - [Read The Isolation and Culture of Rat Hepatic Cells Protocol]

Category: Mouse Protocols: Mouse Cell Culture Protocols

The liver of a rat is cannulated and perfused in situ with buffer, following which it is excised and perfused in a closed system with a collagenase solution. After a period of time the liver begins to break up, at which point it is transferred to a measuring cylinder and culture medium is added. It is then gently agitated to cause the release of cells which are subsequently filtered and allowed to settle out. The parenchymal and non-parenchymal cells form two distinct layers which can be separat - [Read The Isolation and Culture of Rat Hepatic Cells Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

This method enables the isolation of different populations of liver cells and describes their subsequent culture. - [Read The Isolation and Culture of Rat Hepatic Cells Protocol]