preparing Protocols

Category: Immunology: Chemotaxis

12-well Chemotaxis Chamber Protocol. Preparing the Chamber, Preparing and Adding Responding Cells, Removing, Wiping and Staining the Filter. Neuroprobe - [Read 12-well Chemotaxis Chamber Protocol]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

There are two basic methods for the in vitro assay of B-galactosidase activity from yeast. They differ mainly in the method of preparing the material for assay. Both methods are described with accompanying protocols. Method I: Assay of Crude Extracts includes: Yeast Cell Growth; Yeast Cell Harvest; B-gal assays; Bradford Assays. Method II: Permeabilized cell assay. - [Read Assay of β-Galactosidase in Yeast Protocol]

Category: DNA Protocols: cDNA Library Protocols

This stage achieves four goals: polishing the ends of double-stranded DNA, ligation of synthetic linkers or adaptors, digestion of the attached linkers to create cohesive termini, and preparing the cDNA for cloning. - [Read Attachment of Linkers or Adaptors for Construction of cDNA Libraries]

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: Microbiology: Bacterial Competent Cells Preparation E.Coli

Competent cells, calcium chloride method, E. coli, description. CaCl2 and DMSO methods for preparing bacterial and E.Coli competent cells. Zappe, H. BBHelp. - [Read Competent cells, calcium chloride method, E. coli, description]

Category: DNA Protocols: DNA Sequencing Protocols

Direct sequencing from amplified bacterial and large insert cloned human or mouse genomic DNA via an improved MultiPlex PCR-based method. Includes: Preparing primers for MP-PCR; Amplification; PCR Product Clean-up; Sequencing. - [Read Direct Sequencing Using MultiPlex PCR-Based Method]

Category: Bacterial Cell Culture Protocols: Bacterial Cell Culture Media Protocols

This protocol has been used successfully to 15N or 13C/15N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts: - [Read Expression Protocol in M9 Minimal Media via T7 Promoter]

Category: Yeast Protocols: Yeast Genetics Protocols: YACs Protocols

Protocol describes methods for isolation of DNA from a strain of S. cerevisiae carrying a recombinant YAC. Because the linear YAC DNAs are sensitive to shearing forces, pipettes with wide-bore tips should be used to transfer DNAs. The method is suitable for preparing DNA that will be used for agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, PCR, or other methods that do not require intact high-molecular-weight DNA. - [Read Growth of S. cerevisiae and Preparation of DNA Protocol]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Human embryonic stem cells (hESCs) are pluripotent cells capable of differentiation to representatives of all three germ layers. Includes: Isolation of Primary Mouse Embryo Fibroblasts; Thawing and preparing p1 MEF feeder plates; Preparation of MEF- Conditioned Medium (MEF-CM; Microdissection Passaging of hESCs; Bulk passaging of hESC; Cryopreservation of hESCs; Thawing of hESCs; Karyotyping. - [Read Human Embryonic Stem Cell Protocols]

Category: Western Blot Protocols: General Western Blot Methods Protocols

For immunoblotting experiments, it is often important to compare the total amount of an antigen from many different sources or to learn if a particular source has the antigen under study.  In the approach described here, tissue cultures, bacteria, yeast cells, tissues, and other sources of antigens are disrupted directly in an electrophoresis sample . - [Read Immunoblotting: Preparing Cell Lysates Protocol]

Category: Western Blot Protocols

Protein immunoprecipitation can be a useful preparative step for immunoblotting.  For very rare proteins, the protein of interest can be purified and concentrated by standard immunoprecipitation techniques before immunoblotting.  In addition, protein-protein interactions can be tested with an immunoprecipitating antibody that is specific for one protein of a complex and an immunoblotting antibody that is specific for a second member of the complex. - [Read Immunoblotting: Preparing Immunoprecipitated Proteins Protocol]

Category: Western Blot Protocols: General Western Blot Methods Protocols

Protocol describes how proteins are mixed with a sample buffer and prepared for electrophoresis on standard Tris/glycine SDS-polyacrylamide gels. - [Read Immunoblotting: Preparing Protein Solutions Protocol]

Category: Immunology: Immunoprecipitation Protocols

Protocol for immunoprecipitation. Includes: Preparing Cell Lysate; Immunoprecipitating; Western Blotting. - [Read Immunoprecipitation Protocol II]

Category: Immunology: Mononuclear Cell Isolation Protocols

Presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen-presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. - [Read Isolation of Dendritic Cells Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols

Covers techniques for preparing lymphoid cell suspensions and removing red blood cells and dead cells. - [Read Isolation of Mouse Mononuclear Cells Protocol]

Category: Yeast Protocols: Yeast Staining Protocols

Protocol describes our method for preparing cells for immunofluorescence, in which all incubations and washes are performed in microtiter dishes. The protocol can also easily be adapted for preparing cells for immunofluorescence in microfuge tubes. - [Read Large-Scale Immunocytology Protocol]

Category: Histology Protocols: Laser Capture and Microdissection

Preparation of Frozen Tissue for LCM, Preparing Sections from Frozen Tissue for Laser Microdissection, Preparing Sections from Fixed, Paraffin-embedded Tissue for Laser Microdissection Protocols and methods. Laser Microdissection Laboratory. UAB. - [Read Laser Capture Microdissection Protocols]

Category: C. elegans Protocols: C. elegans Cell Culture Protocols

Protocol for liquid culture of worms. Includes: superbroth; S- basal; worm plates; GROWING THE BACTERIA (WORM FOOD); GROWING THE WORMS; AFTER THE CULTURE HAS GROWN; PREPARING EGGS TO START SYNCHRONIZED LIQUID CULTURES. - [Read Liquid Culture of Worms Protocol]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Caenorhabditis elegans, a small (adults are ~1 mm long), free-living soil nematode that feeds on bacteria, is an ideal organism for applying various live microscopy techniques. This protocol describes useful techniques for preparing C. elegans for live microscopic analysis. Details of sample preparation depend on the developmental stage of the worm to be studied. - [Read Live Imaging of Caenorhabditis elegans: Preparation of Samples]

Category: C. elegans Protocols

Protocol describes useful techniques for preparing C. elegans for live microscopic analysis. Details of sample preparation depend on the developmental stage of the worm to be studied. - [Read Live Imaging of Caenorhabditis elegans: Preparation of Samples]

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

This protocol gives instructions for preparing microsomes from a rat liver. - [Read Microsome Purification from Rat Liver Protocol]

Category: Fungi Protocols: Neurospora Protocols

Northern blot protocol for the detection of RNA in Neurospora. Includes: Extract RNA from tissue powder; Electropheresis and transfer RNA; Preparing the membrane for probing and preparation of a riboprob.; Hybridization. - [Read Northern blot Protocol for the Detection of RNA in Neurospora]

Category: Lipid Protocols

Protocol for the preparation of bDOG-DOPG mixed micelles. Includes: Recrystallizing BOG; Preparing mixed micelles. - [Read Preparation of bOG-DOPG Mixed Micelles Protocol]

Category: Nucleic Acid Detection Protocols

Protocol for preparation of fluorescent DNA probe from HUMAN mRNA or total RNA using direct incorporation. Includes: Preparing fluoresenctly labeled cDNA (probe); Washing and Scanning Arrays. - [Read Preparation of Fluorescent DNA Probe from HUMAN mRNA or Total RNA using Direct Incorporation]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol for the preparation of fluorescent DNA probe from HUMAN mRNA or total RNA using direct incorporation. Includes: Preparing fluoresenctly labeled cDNA (probe); Washing and Scanning Arrays. - [Read Preparation of Fluorescent DNA Probe from HUMAN mRNA/ Total RNA using Direct Incorporation Protocol]