single Protocols

Category: Fungi Protocols: Aspergillus Protocols

The technique makes use of an Escherichia coli strain expressing the redΑßΓ operon under the control of an inducible promoter. This enables the strain to carry out homologous recombination with only 50-60 bp of homologous sequence. The procedure does not require any DNA ligation and is very rapid. It allows a single gene or region on a cosmid to be replaced by a bi-functional selectable marker (having both an E. coli and an A. fumigatus marker). - [Read A Rapid Method for Generating Gene Deletions in Aspergillus fumigatus Protocol]

Category: Cell Biology and Cell Culture: Apoptosis Protocols

A sensitive method for detection of apoptosis by single laser Flow Cytometry. - [Read A sensitive method for detection of apoptosis by single laser FACS]

Category: Nucleic Acid Purification Protocols

Protocol for a single-step method for the simultaneous preparation of DNA, RNA, and protein from cells and tissues. The yield of total RNA depends on the tissue or cell source, but it is generally in the range of 4-7 µg/mg starting tissue or 5-10 µg/106 cells.  IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. - [Read A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissue]

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

Describes an experimental cross in mice that can be used to define and map induced germ-line mutations that map to a single chromosome. The cross is a modification and extension of a conventional three-generation recessive mutagenesis screen. Includes: The Mutagenesis Breeding Plan; Consomic Strains; Generating Mutations; Generating and Genotyping G2 Females; Genotyping G3 Progeny; Phenotyping G4 Progeny; etc.. - [Read A Targeted Screen to Detect Recessive Mutations that have Quantitative Effects Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses a single thermostable RNA polymerase to perform high-specificity RT-PCR.  A high-temperature RT reaction is followed by PCR amplification of the cDNA using a single thermostable poymerase, the GeneAmp AccRT RNA PCR enzyme from Applied Biosystems.  The high temperature of the RT reaction enhances the specificity of primer binding and also reduces secondary structure in the template, thereby increasing the efficiency of polymerization. - [Read Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium]

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

An Integrative Procedure for Apoptosis Identification and Measurement. Yingyu Cui Lab/Group: National Key Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences. I have uploaded an integrative procedure through which a relatively satisfactory result can be obtained following a single stage of cell culture and transient cell treatment, then detection with different instruments. This shortens experiment time. - [Read An Integrative Procedure For Apoptosis Identification And Measurement]

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

Provides several approaches to flow cytometry data analysis. Frequency determinations based on analysis of single-parameter fluorescence histograms and dual-parameter contour plots are presented. Steps are described for calculating values for signal-to-noise ratios when logarithmic amplification is used for data collection. - [Read Analysis of Flow Cytometry Data Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

A rapid method to analyze the size of the single-stranded DNA of M13 recombinants. - [Read Analysis of Recombinant Bacteriophage M13 Clones Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

A sensitive method for the detection of apoptosis by single laser flow cytometry. Methodology includes: Staining for detection of apoptosis, Direct Staining Procedure, Indirect Staining Procedure, Protocol for the use of actinomycin D (AD) on samples that were stained with 7-AAD for apoptosis and fixed in formaldehyde. - [Read Apoptosis Detection Protocol By Single Laser Flow Cytometry]

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

An integrative procedure through which a relatively satisfactory result can be obtained following a single stage of cell culture and transient cell treatment, then detection with different instruments. This shortens experiment time. - [Read Apoptosis Identification and Measurement Protocol]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

This assay is used when working with phage vectors carrying the beta-gal gene. If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-gal gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta-gal will work as indicator host bacteria; a single chromosomal copy of the gene is not a problem. - [Read Assay for Phage Containing the Beta-galactosidase Gene]

Category: Signalling Signal Transduction Protocols

Assay of cytokines in tissue culture supernatants describes a liquid suspension array for quantification of cytokines in tissue culture supernatants or serum. With this assay, it is possible to profile the level of multiple cytokines in a single well. The principle of this cytokine assay is similar to a capture sandwich immunoassay. Includes: Preparation for the Assay, Cytokine Assay, Reagents and Materials. - [Read Assay of Cytokines in Tissue Culture Supernatants]

Category: Immunology: Immunoassays

The Bio-Plex cytokine assay employs a liquid suspension array for quantification of cytokines in tissue culture supernatants or serum. Using this 96-well microtiter plateformatted assay, it is possible to profile the level of multiple cytokines in a single well. - [Read Assay of Cytokines in Tissue Culture Supernatants Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

This protocol describes an in vitro transcription assay that allows for a single round of transcription from in vitro assembled chromatin. Comparing the activity of a receptor or transcriptional coactivator in an assay that measures only a single round of transcription with the results from multiple rounds of transcription can help elucidate the mechanism of transcriptional activation by those factors. - [Read Assay:Single Round of In Vitro Transcription from Assembled Chromatin Templates Using a HeLa Cell Ex]

Category: Cloning Protocols

Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms. - [Read Attaching Adaptors to Protruding Termini Protocol]

Category: C. elegans Protocols: C. elegans Genetics Protocols

The procedure is to mutagenize a large population of worms with trimethylpsoralen and UV irradiation, set up 1152 subpopulations, screen DNA made from this library for deletions in specific genes by nested PCR, and then to recover single worms carrying the deletions through a sib-selection process. - [Read C. elegans Gene Knockout Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Choosing the right labeling method for your hybridization experiment. Includes: Homogeneous labeling methods for DNA; Homogeneous labeling methods for RNA; Stability of probe-target interaction; Nonradioactive labeling of oligonucleotides; Double-stranded versus single-stranded probes. - [Read Choosing the Right Labeling Method for your Hybridization Experiment]

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: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol describes three standard methods to construct bacteriophage M13 recombinants: (1) ligating insert DNA to a linearized vector, prepared by cleavage of M13 RF with a single restriction enzyme; (2) using alkaline phosphatase to suppress self-ligation of the linearized vector, and (3) using M13 RF cleaved with two restriction enzymes for directional cloning. - [Read Cloning into Bacteriophage M13 Vectors Protocol]

Category: DNA Protocols: DNA Sequencing Protocols

Protocol first describes the vector preparation and, then, describes the insert preparation.  Vital to have an excellent vector in order to produce a sequencing library.  Protocol employs the male-specific coliphage M13 as the sequencing vector.  M13 is a filamentous phage with a single-stranded, circular genome.  M13 is widely used as a vector because many versions are available commercially and because M13 has certain advantages. - [Read Construction of the Sequencing Library Protocol]

Category: Cytogenetics Protocols

CGH provides a comprehensive picture of all chromosomal gains and losses within a single experiment. In contrast to banding analysis, CGH requires no preparation of metaphase chromosomes from the cells to be analyzed (which in certain tumor types may be difficult or even impossible). - [Read Detection of Chromosomal Imbalances Using DOP-PCR and Comparative Genomic Hybridization (CGH)]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

The most convenient and commonly used method to visualize DNA in agarose gels is staining with the fluorescent dye ethidium bromide.  Ethidium bromide can be used to detect both singleand double-stranded nucleic acids (both DNA and RNA).  However, the affinity of the dye for single-stranded nucleic acid is relatively low and the fluorescent yield is comparatively poor. - [Read Detection of DNA in Agarose Gels Protocol]

Category: Genetics and Genomics: Cancer Genetics Protocols: Single Strand Conformational Polymorphism(SSCP) Protocols

Single-strand conformational polymorphism (SSCP), one of several methods used to scan segments of DNA for mutations, exploits the electrophoretic differences in mobilities between single-stranded mutant and wild-type DNAs. - [Read Detection of Mutations by Single-strand Conformational Polymorphism Protocol]

Category: Fungi Protocols: Fungi Genetics Protocols

Standard operating procedure for the determination of tissue fungal burden utilizing quantitative real time polymerase chain reaction (QPCR). This standard operating procedure will provide information on how to assess fungal tissue burden of infected animals by use of a single copy (FKS) or multicopy gene (18s RNA) to assess the number of fungal cell nuclei present. - [Read Determination of Tissue Fungal Burden Utilizing Quantitative Real Time Polymerase Chain Reaction]

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

Protocol describes DNA-sequencing of single-stranded DNA templates in reactions catalyzed by Sequenase. - [Read Dideoxy-mediated Sequencing Reactions Using Bacteriophage T7 DNA Polymerase (Sequenase) Protocol]