general Protocols

Category: Immunohistochemistry Protocols: Fixation Protocols

Information on histotechniques. Includes: Fixation - types of fixatives; factors affecting fixation; General usage of fixatives; Tissue Processing; Sectioning; Frozen Sections; Staining; H and E staining; Coverslipping; Decalcification; Artefacts in Histologic Sections; Problems in Tissue Processing. - [Read Histotechniques Fixation]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocols and information on general HPLC column care. Includes: Silica based columns and Polymer based columns.  Protocols included: pH stability, Mechanical stability, Mobile phases (Eluents), Proper storage of HPLC columns, Equilibration time, Regeneration of a column, Regeneration of RP packings, Regeneration of NP (Normal Phase) packings, Regeneration of Ion Exchange Packings - [Read HPLC Column Care Protocols and Information]

Category: HPLC Protocols

HPLC Protocol. A general HPLC procedure to get you started on High Performance Liquid Chromatography. - [Read HPLC Protocol]

Category: DNA Microarray Protocols

Hybridization of Arrays. . General/Yeast Microarrays. Brown Lab - [Read Hybridization of Arrays]

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: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol outlines the general procedure and requirements for in vitro translation of CFTR and outlines some assays using in vitro translated product. Core glycosylation of CFTR occurs in the ER. An assay for this processing step requires the addition of microsomal membranes to the basic in vitro translation mixture. This protocol takes this into account. - [Read In vitro Translation Assays for CFTR]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: Long PCR Protocols

General Guidelines for Long PCR Conditions and Enzyme Mixtures. Pete Estep. Harvard. - [Read Long PCR Reagents and Guidelines]

Category: PCR Protocols: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

This simple cell culture-based cytotoxicity test (in which cell viability is determined by uptake of the dyes ethidium bromide and fluorescein acetate) has been developed as a general test for acute toxicity. - [Read LS-L929 Cytotoxcitiy Test]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Several common drugs, their targets, and protocols are described for studying organelle distribution and trafficking. The drugs are readily available from general suppliers, including Sigma, Roche, and Calbiochem. - [Read Membrane Trafficking and Organelle Reagents]

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

Human tissues are comprised of multiple interacting cell populations in a complex three dimensional arrangement with each cellular phenotype determined by a unique profile of mRNA and protein expression. Before microdissection techniques were developed, the only analysis tools for phenotypic studies were primarily immunohistochemistry and in-situ hybridization. While useful, these tools are limited to single gene analysis and, in general, do not allow qualitative studies. - [Read Microdissection Overview]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture

Boris Greber. Important rules for ES cell culture, Signs of differentiation, General tips, ES medium, Thawing ES cells, Freezing, Passaging. - [Read Mouse embryonic stem (ES) cell culture - basic procedures]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

For both biological and economical reasons, it is important to eliminate mycoplasmas from cell cultures being used for basic research, diagnosis, and biotechnological production. The most commonly used method for elimination, inactivation, or suppression of mycoplasmas in cell cultures is treatment with antibiotics. In general, antibiotic therapies do not result in long-lasting, successful elimination. Also, the cytotoxic properties of antibiotics can cause undesirable side effects on cells. - [Read Mycoplasma Elimination Reagent Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Discusses the effects of various components of the hybridization solution on the rate of renaturation and thermal stability of DNA hybrids free in solution. Includes: The main parameters that influence hybridization; Additional hybridization variables; Competition in situ hybridization; Oligonucleotide hybridization; Standard in situ hybridization conditions. - [Read Nucleic Acid Hybridization General Aspects]

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol describes transfection of plasmid DNA into mammalian cell lines using electroporation, a process whereby external application of electric pulses induce cell membrane permeability. A number of factors can affect electrotransfection efficiency. In general, cells in suspension and small volume cells are difficult to transfect, whereas adherent cells and large volume cells are relatively easy. - [Read Optimizing Electrotransfection of Mammalian Cells In Vitro Protocol]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: PCR Optimization Protocols

PCR Tips and Tricks - General Guidelines and Troubleshooting PCR polymerase chain reaction. The Genomic Variation Laboratory, U.C Davis - [Read PCR Tips and Tricks]

Category: PCR Protocols: PCR Optimization

PCR Tips and Tricks - General Guidelines and Troubleshooting PCR polymerase chain reaction. The Genomic Variation Laboratory, U.C Davis - [Read PCR Tips and Tricks]

Category: Peptide Protocols: Peptide Storage

In general, peptide solutions are stable for up to a week at 4°C. However, if the peptide sequence has inherent instability (Peptide Stability), it might be better to freeze the solution when not in use. Peptide solutions at pH>8 should also be frozen when not in use. Sigma Aldrich. - [Read Peptide Handling and Storage]

Category: Peptide Protocols: Dissolving Handling Peptides

Peptide Handling Guides. Storage Guidelines for Lyophilized Peptides, Strategy for Dissolving Single Peptides, Determining Solubility Characteristics of peptides, Dissolving Approach for Charged Peptides, Dissolving Approach for Hydrophobic/Uncharged Peptides, Guidelines for Dissolving Several Peptides, Peptide Stability and Potential Degradation Pathways, and storage of peptides. Sigma Aldrich. PDF. - [Read Peptide Handling Guides]

Category: Viral Manipulation Protocols: Phage Protocols

General method to pick and store plaques. - [Read Picking Bacteriophage lambda Plaques Protocol]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol describes a general approach for the direct attachment of a triazine dye onto a carbohydrate support.  The direct coupling method is achieved under alkaline conditions (pH 10-11) with nucleophilic displacement of the dye’s chlorine atom by the polymer hydroxyls. - [Read Preparation of Affinity-Ligand Resins by Immobilization of Dyes on Polyhydroxyl Matrices Protocol]

Category: Animal Research Techniques

Legal responsibilities, Recognition and treatment of pain, Anesthetic monitoring, stages and planes of general anesthesia, injecting, inhalation, species-specific, anesthetic emergencies, controlled substances. Marilyn J. Brown UCSB - [Read Principles of Anesthesia and Analgesia: ESSENTIALS FOR ANIMAL RESEARCH]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Immunoaffinity purification of antibodies is used to purify antigen-specific antibodies from a preparation of polyclonal antibodies.  Such purification is commonly needed in the production of antipeptide antibodies, where it is used to concentrate the desired antibodies and separate them from those raised against carrier proteins.  It is also used for the more general purpose of removing unwanted, nonspecific binding activity from polyclonal antibody preparations. - [Read Purification of Antibodies on an Antigen Column Protocol]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol describes a general method for the purification of protein using dye-ligand affinity chromatography.  The effectiveness of this protocol is dependent upon the reader having previously determined the optimal dye and buffer conditions to use for binding and eluting the protein(s) of interest. - [Read Purification of Protein Using Dye-Ligand Affinity Chromatography Protocol]

Category: ELISA Protocols: Sandwich ELISA Assay Protocols

Sandwich ELISA Protocol. Chemicon. General Protocol for the Sandwich ELISA method, Troubleshooting Sandiwich ELISA Assays. - [Read Sandwich ELISA Protocol]