conventional Protocols

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: Molecular Biology Protocols: Carbohydrate Protocols

High performance liquid affinity chromatography (HPLAC) is a useful procedure to investigate he interactions between carbohydrate binding protein and their ligands. Technical requirements are similar to conventional HPLC. HPLAC can screen and separate natural ligands from complex biological mixtures. WeiTong Wang~GlycoTech Corporation, Rockville, Maryland - [Read Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography]

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

The ability to synthesize RNA in the lab is critical to many techniques.Radiolabeled and nonisotopically labeled RNA probes, generated in small scale transcription reactions can be used in blot hybridizations and nuclease protection assays. This article includes information on: Requirements For Transcription, RNA Phage Polymerases, Template Options: Plasmids, PCR Products, Oligonuclotides and cDNA, Sense or Antisense, Conventional Or Large Scale Synthesis, Products for In Vitro Transcription. - [Read Basic Information on In Vitro Transcription]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol for configuration, column construction, and column packing for a capillary liquid chromatography system. Protocol describes a procedure for adapting conventional HPLC systems to provide accurate low-flow rates (0.4-4 µl/min) and gradients required to operate slurry-packed capillary columns.  A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to several commercial UV detectors. - [Read Configuration Column Construction Column Packing for Capillary Liquid Chromatography]

Category: Microscopy Protocols: Optical Microscopy Protocols

The visibility of the faint star light is enormously enhanced against a dark background. This principle is applied in darkfield (also called darkground) microscopy, a simple and popular method for making unstained transparent specimens clearly visible. Such objects often have refractive indices very close in value to that of their surroundings and are difficult to image in conventional brightfield microscopy. - [Read Darkfield Illumination]

Category: Molecular Imaging: Confocal Microscopy Protocols and Information

General Confocal Protocols. Confocal Startup Protocol, Confocal Clean-Up Protocol, Confocal Printing Protocol.CONVENTIONAL MICROSCOPY NOTES. Piscataway, New Jersey. Rutgers University - [Read General Confocal Protocols]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Labeling Protocols

Hybridization is carried out in conventional aqueous solvents at a temperature well below the predicted melting temperature.  Nonspecific hybrids are then removed by washing at high stringency in buffers containing quaternary salts.  Tetramethylammonium chloride (TMACl) is used with probes that are 14-50 nucleotides in length, whereas tetraethylammonium chloride (TEACl) is used with longer oligonucleotides. - [Read Hybridization of Oligonucleotide Probes in Aqueous Solutions Protocol]

Category: Antibody Protocols: Antibody Labeling Protocols

This method for tagging monoclonal antibodies involves growing hybridomas in the presence of radioactive amino acids. This protocol can be particularly useful when conventional labeling techniques cause the antibody to lose activity. The labeled antibodies that result are essentially identical to the unlabeled antibodies. - [Read Labeling Monoclonal Antibodies by Biosynthesis Protocol]

Category: C. elegans Protocols

To image early cleavages and chromatin dynamics, it is convenient to use histone H2B fused to GFP or lamin::GFP. Time-lapse movies can be obtained using conventional confocal microscope systems and their included software. Early embryos dissected from transgenic hermaphrodites are placed with egg salts on agar pads. - [Read Live Imaging of Caenorhabditis elegans: Examples]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol describes a procedure for adapting conventional HPLC systems to provide accurate low-flow rates (0.4-4 µl/min) and gradients required to operate slurry-packed capillary columns.  A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to a number of commercial UV detectors. - [Read Packing Capillary Columns for RP-HPLC Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for preparation of DNA for pulsed-field gel electrophoresis: isolation of intact DNA from yeast. Yeast cells are first treated enzymatically to break down the cell walls and then resuspended in low-melting-temperature agarose plugs.  The DNA is liberated by infusing the plugs with lysis buffer and proteases.  This method is used to prepare both conventional and artificial yeast chromosomes. - [Read Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of Intact DNA from Yeast]

Category: Immunology: T Cell Protocols

Protocols describe the conditions required to induce proliferation are described. Also describe the assay in which CD4·CD25·T cells are co-cultured with conventional T cells in order to assess their suppressive function. Will describe the culture conditions for the activation and expansion of CD4·CD25· cells. - [Read Proliferative Assays for T Cell Function Protocols]

Category: Microscopy Protocols: In Vivo Imaging Protocols

To image early cleavages and chromatin dynamics, it is convenient to use histone H2B fused to GFP or lamin::GFP. Time-lapse movies can be obtained using conventional confocal microscope systems and their included software. Early embryos dissected from transgenic hermaphrodites are placed with egg salts on agar pads. Chromatin dynamics can be followed easily, and wild-type embryonic cells can be compared with mutants or RNAi-treated embryos. - [Read Protocol Live Imaging of Caenorhabditis Elegans]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this protocol, double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]

Category: PCR Protocols

Protocol describes how double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

This procedure, which uses a root transformation protocol, provides a rapid method for assessing gene expression in Arabidopsis roots. It is useful for testing promoter:reporter gene constructs, for expressing genes, the overexpression of which is lethal in whole plants, and for transforming the roots of plants that are recalcitrant to conventional transformation techniques. The protocol has been used successfully with Ws, No-0, and RLD ecotypes. - [Read Transgene Expression in Regenerated Roots]