individual Protocols

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

This protocol provides a method for analysis of DNA fragmentation using flow cytometry after propidium iodide (PI) labeling of apoptotic nuclei. Apoptotic nuclei stain less than their normal counterparts because of diffusion of low-molecular-weight fragments out of the cells and/or chromatin condensation. - [Read Analysis of DNA Fragmentation Using Propidium Iodide (PI) Fluorescence of Individual Nuclei Protocol]

Category: Neuroscience Protocols: Patch Clamping Protocols

Combined patch clamp recording and mRNA expression profiling in individual neurons. - [Read Combined Patch Clamp Recording and mRNA Expression Profiling in Individual Neurons]

Category: Microscopy Protocols: Optical Microscopy Protocols

When imaging specimens in the optical microscope, differences in intensity and/or color create image contrast, which allows individual features and details of the specimen to become visible. Contrast is defined as the difference in light intensity between the image and the adjacent background relative to the overall background intensity. In general, a minimum contrast value of 0.02 (2 percent) is needed by the human eye to distinguish differences between the image and its background. - [Read Contrast in Optical Microscopy]

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol describes a method for disaggregating embryos and the ICM of blastocysts into individual cells. - [Read Disaggregating Cleavage-Stage Embryos and the Inner Cell Mass of Blastocysts into Individual Cells]

Category: Drosophila Protocols: Drosophila Nucleic Acids Protocols

Protocol for Drosophila single-fly DNA preps for PCR. Includes: DNA Preparation Protocol; DNA Preps from many individual flies; Inverse PCR. - [Read Drosophila Single-Fly DNA Preps For PCR Protocol]

Category: Cell Culture Protocols

Protocol describes a method for estimation of mammalian cell number in a defined volume of medium using a hemocytometer. Automated methods using cell-counting devices such as those produced by Coulter are desirable when large numbers of individual samples are to be counted. - [Read Estimation of Cell Number by Hemocytometry Counting Protocol]

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

Flow cytometry is a widely used method for characterizing and separating individual cells. This basic protocol focuses on: measure fluorescence intensity produced by fluorescent-labled antibodies and ligands that bind specific cell-associated molecules. Includes: Immunofluorescence Staining and Flow Cytometry Analysis. - [Read Flow Cytometry Analysis Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Flow cytometry is a widely used method for characterizing and separating individual cells. This basic protocol focuses on: measure fluorescence intensity produced by fluorescent-labled antibodies and ligands that bind specific cell-associated molecules. Includes: Immunofluorescence Staining; Flow Cytometry Analysis. - [Read Flow Cytometry Analysis Protocol]

Category: Chromatography Protocols

FPLC Protocol. The FPLC consists of a pump and a column which will withstand high pressure so separations can be carried out relatively quickly. For a detailed description there is a FPLC system handbook which is particularly useful for trouble shooting. For use of individual columns follow the "instructions" (in the green folder) which accompany each one. Sir William Dunn School of Pathology, Oxford University. - [Read FPLC Protocol]

Category: Signalling Signal Transduction Protocols

This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography. Includes information: H1 Kinase Assay on Individual Xenopus Oocytes; H1 Kinase Assay on Xenopus Egg Extract Samples; H1 Kinase Assay on Tissue Culture Cells; Helpful protocol hints. - [Read Histone H1 Kinase Activity Assay]

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

Useful techniques to circumvent disruption of tissue structure in the analysis of gene expression are LCM and LDM. While they require specialized microscopes and systems, they are similar in that freshly-cut frozen tissue sections can be microdissected using either a general histological stain (like H&E) or by staining with fluorescently conjugated antibodies. The LCM system by Arcturus involves... - [Read Immunofluorescent Staining for the Laser Microdissection of Individual Cells Protocol]

Category: Signalling Signal Transduction Protocols: Cytokine Protocols

Fluorescent anti-cytokine and monoclonal antibodies are useful for the intracellular staining and multiparameter flow cytometric analysis of individual cytokine-producing cells within mixed cell populations. BD Biosciences. - [Read Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis]

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: PCR Protocols: Inverse PCR Protocols

Inverse PCR is used to amplify and clone unknown DNA that flanks one end of a known DNA sequence and for which no primers are available.  The technique involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region.  The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in the PCR. - [Read Inverse PCR Protocol II]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

This protocol describes a method for isolating individual ES cell colonies by picking after they have grown on selective medium. - [Read Isolating Individual Embryonic Stem (ES) Cell Colonies by Picking Protocol]

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: Protein Protocols: Protein Quantitation Concentration Protocols

Find a list of assays for the determination of protein concentration in a solution.  This list includes the sensitivity range, volume/amount of sample needed, subjective comments on accuracy and convenience, and major interfering agents.  Procedural details, equipment requirements, and references are outlined in the individual assay documents. - [Read List of Protein Assays]

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

Microneedles attached to micromanipulators are used in the dissection of tetrads, isolation of zygotes from populations of mating haploid cells, and manipulation of individual cells. - [Read Making a Tetrad Dissection Needle Protocol]

Category: PCR Protocols: Colony PCR

The following colony PCR protocol has been designed to be performed in individual reaction tubes. We usually test three colonies from each transformation along with a wild-type control. A series of five different PCR tests are performed on each colony to - [Read Single tube confirmation PCR protocol]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes how to prepare double-stranded RNA (dsRNA) for RNA interference in Drosophila by synthesis of individual RNA strands from linearized plasmid templates, followed by annealing of the strands.  DsRNA molecules with a length of 500-800 bp seem to be most active.  The dsRNA can be made from cDNA or genomic DNA templates, as long as most of the dsRNA corresponds to presumptive exon sequence. - [Read Synthesis of dsRNA for RNAi in Drosophila: Plasmid Template Method Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

When an individual bacterial virus grows in a bacterial host suspended in a top agar lawn, its progeny infect and lyse the surrounding host cells. This causes the appearance of a "hole" or plaque in the otherwise homogeneous bacterial lawn. Since each plaque represents a single virus, the number of viruses in the aliquot added to the plate is equal to the number of plaques which appear. - [Read Titering of Bacterial Viruses Protocol]

Category: Plant Biology Protocols

Alfalfa is an outcrossing species, cultivars consist of populations rather than individual homozygous or inbred lines. After only two cycles of self-pollination, severe inbreeding depression eliminates selfed individuals from populations. To obtain sufficient plant material of relative genetically uniformity (these plants will still necessarily be heterozygous) for experimental purposes, it may be necessary to propagate a single individual or clone. - [Read Vegetative Propagation of Alfalfa by Stem Cuttings]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

During development many plant cells undergo endoreduplication, whereby ploidy increases to a multiple of the normal 2C content. For eg., trichome development is accompanied by an increase in ploidy to 32C, indicating that trichome cells undergo four rounds of endoreduplication. Protocol describes DNA levels, and hence developmental progress in the corresponding cells, are measured by staining the DNA with a fluorescent marker and then quantifying the fluorescence of individual nuclei. - [Read Whole-Mount DAPI Staining and Measurement of DNA Content in Plant Cells]