approach Protocols

Category: Molecular Biology Protocols: DNA Ligation Protocols

Addition of linkers/adaptors to blunt-end DNA fragments. Ligation of mixture containing DNA and linkers/adaptors. Powell Gannon. Oxford Practical Approach Series. - [Read Addition of linkers/adaptors to blunt-end DNA fragments PDF]

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

Aggregation of ES Cells with Mouse Morula-state Embryos. Oxford Practical Series Approach. - [Read Aggregation of ES Cells with Mouse Morula-state Embryos]

Category: Neuroscience Protocols

After section of the corticospinal (CS) tract in adult rats, neutralizing Nogo-A with monoclonal antibodies leads to enhancement of axonal re-growth and compensatory sprouting, accompanied by increased motor recovery.  The neutralization of Nogo-A represents a promising approach for therapy after lesion if its enhancement of functional recovery can be transposed to primates. - [Read Anti-Nogo-A Treatment Protocol]

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Protocol describes the use of chromatin immunoprecipitation technology (ChIP) to analyze interactions of proteins or protein complexes with DNA in vivo. In this approach, the material is fixed with formaldehyde to preserve DNA-protein and protein-protein associations, the cells are lysed, and the chromatin is cut and solubilized. The chromatin suspension is immunoprecipitated with an antibody against the protein(s) of interest, and the coimmunoprecipitated DNA fragments are analyzed. - [Read Chromatin Immunoprecipitation (ChIP) of Protein Complexes Protocol]

Category: Transgenic Mouse Protocols: Mouse Genotyping Protocols

Practical Approach Online, Oxford University Press. Combined LacZ staining and in situ hybridization of mouse embryos, beta-galactosidase. - [Read Combined LacZ staining and in situ hybridization PDF]

Category: Cloning Protocols: Mutagenesis Protocols

This approach can be used to delete a fragment of any lenth or to introduce point mutations into a DNA seqence. - [Read Creating a Deletion by PCR Splicing Protocol]

Category: Molecular Biology Protocols: DNA Ligation Protocols

DNA ligation Method, Equipment and reagents, T4 DNA ligase. Gannon and Powell. Oxford Practical Approach Series. - [Read DNA Ligation Protocol PDF]

Category: Microinjection Protocols: Microinjection of DNA Protocols

For microinjection into cells, high-quality plasmid DNA is purified using standard procedures. The resulting preparation is then delivered into the microinjection needle by either a back-filling or a forward-filling approach. - [Read DNA Preparation and Needle Loading for Gene Delivery by Microinjection Protocol]

Category: Immunology: B Cell Protocols

B cell activation can be quantitated indirectly by assaying antibody production or directly by measuring cellular changes that occur immediately after exposure to an activation signal. Provides methods for the latter (direct) approach--namely, methods for quantifying early parameters of B cell activation such as increases in intracellular ionized calcium concentration [Ca2+]I, cell size, and MHC class II-antigen expression. - [Read Early Events in B Lymphocyte Activation Protocol]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: ES Cell Growth Media and Requirements

Reference: Michael P. Matise, Wotjek Auerbach and Alexandra L. Joyner (2000). Gene targeting: a practical approach. Protocol excerpted from Chapter 3, Production of targeted embryonic stem cell clones. Alexandra L. Joyner (ed.), 2nd edition, Oxford Unive - [Read Embryonic Stem Cell growth media Requirements - Taconic Transgenics]

Category: Immunology: Immunoprecipitation Protocols

Protocols for detection and purification of proteins tagged with a particular epitope, the FLAG tag, although the same general approach can be applied to other epitope tags. The protocols employ the anti-FLAG M2 antibody to detect and purify FLAG-tagged proteins. The methods presented are immunoprecipitation of FLAG fusion proteins from cells using an anti-FLAG M2 affinity gel, detection of FLAG fusion proteins by western blotting, and purification of FLAG fusion proteins by anti-FLAG. - [Read Epitope Tagging of Recombinant Proteins Protocol]

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

ES Cell Injection Into Mouse Embryos. Practical Approach Online, Oxford University Press. - [Read ES Cell Injection Into Mouse Embryos]

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

Injection of ES Cells Into Morulae. Practical Approach Online, Oxford University Press - [Read Injection of ES Cells Into Morulae]

Category: Immunology: T Cell Protocols

Describes methods for labeling high or low numbers of lymphocytes with CSFE. Protocols are provided to use CSFE-labeled cells in cell transfer studies or as cells to be cultured in vitro. Detailed guidelines for positioning of CSFE-labeled lymphocytes in lymphoid organs or other tissues are included for those wishing to use this approach to study lymphocyte migration. - [Read Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation]

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

Protocol describes an efficient approach to isolate RNA from Caenorhabditis elegans. - [Read Isolation of RNA from C. elegans Protocol]

Category: Molecular Biology Protocols: DNA Ligation Protocols

10 × tailing buffer. Tdt procedure. Terminal transferase and cloning. Powell and Gannon. Oxford Practical Approach Series. - [Read Ligation of DNA fragments by homopolymer tailing]

Category: DNA Microarray Protocols

Making a scanning array: in situ synthesis of oligonucleotides on a solid substrate. Elder et al., Oxford Practical Approach Series. - [Read Making a scanning array: in situ synthesis of oligonucleotides on a solid substrate]

Category: Bacteria Genetics Protocols

Protocol describes a method to determine the presence of plasmid DNA in an Agrobacterium culture. Compared to selection of transformed Agrobacterium, which can be ambiguous and normally takes several days for resistant colonies to appear, the approach described here is both rapid and accurate. - [Read PCR Analysis of Agrobacterium Protocol]

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: Molecular Biology Protocols: Restriction Enzyme Digestion

Sequential digestion of DNA using two restriction endonucleases. Gannon and Powell. Oxford Practical Approach Series. - [Read Sequential digestion of DNA using two restriction endonucleases]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for small marker chromosome identification in metaphase and interphase using centromeric multiplex FISH (CM-FISH). This approach allows the identification of marker chromosomes and other aneuploides on one single cytogenetic slide, in less than two hours. - [Read Small Marker Chromosome Identification in Metaphase and Interphase using Centromeric Multiplex FISH]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Describes T cell enrichment using cytotoxic antibodies, and also describes the depletion of T cells and their subpopulations using the same approach. In the latter unit, T cell surface markers (Thy-1, CD4, and CD8) are targeted by the cytotoxic antibodies. - [Read T Cell Enrichment by Cytotoxic Elimination of B Cells and Accessory Cells Protocol]

Category: Electrophysiology Ion Channels

The Whole-Cell Patch-Clamp Technique – a powerful tool to approach CFTR function. Martin J. Hug. University of Muenster, Institute of Physiology. Germany - [Read The Whole-Cell Patch-Clamp Technique – a powerful tool to approach CFTR function]

Category: Immunohistochemistry Protocols: Antigen Retrieval Protocols

The principle behind the pressure cooker method described here is to use extended periods of heat to break some of the subcellular structures that block antibody access. This approach is appropriate for handling specimens on glass slides. The major advantages of the pressure cooker method are the ability to handle a large number of slides simultaneously, the convenience of using metal racks, and the avoidance of any hot spots that are found in the microwave. - [Read Unmasking Hidden Epitopes Using the Pressure Cooker Protocol]