addition 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: Cytogenetics Protocols

Protocol for Agrobacterium-mediated transformation in rice. Agrobacterium-mediated rice transformation method that is applicable to easily cultured varieties in addition to elite japonica varieties that are more difficult to culture. Using this method, transgenic rice plants can be obtained in about 2–3 months with a transformation frequency of 30–50%, both in easily cultured varieties and recalcitrant elite japonica rice. - [Read Agrobacterium-Mediated Transformation in Rice Protocol]

Category: Protein Protocols: Protein Phosphorylation Protocols

Paper describing methods for monitoring kinase activity, investigating kinase–substrate specificity, examining phosphorylation in planta and the determination of phosphorylation sites in a protein. In addition, strategic considerations for experimental design and variables will be discussed. Scott C. Peck, the Plant Journal. - [Read Analysis of protein phosphorylation: methods and strategies PDF]

Category: Protein Protocols: Immunoprecipitation Protocols

Antibody-antigen complexes are removed from solution by addition of an insoluble form of an antibody binding protein such as Protein A, Protein G or second antibody. Immunoprecipitation protocols / methodology and technical background information. P.J. Ha - [Read Analysis of Proteins by Immunoprecipitation]

Category: C. elegans Protocols: C. elegans Staining Protocols

Extreme care should be used to identify and verify positive reactions, however, because cross-reactions are common. Counterstaining is essential for examining worms by immunofluorescence and is used to identify the exact cell in which an antigen appears. Methods for counterstaining include labeling all cells with a fluorescent dye that is specific for nucleic acids (e.g., DAPI or propidium iodide) and using GFP driven by tissue-specific promoters. - [Read Antibody Addition and Detection for Staining Caenorhabditis elegans Protocol]

Category: Drosophila Protocols

Enzyme-linked reagents give excellent sensitivity and use a simple light microscope for detection. A range of enzymes is available, but for staining in situ, horseradish peroxidase will suit most needs. Diaminobenzidine (DAB) is one of the most sensitive substrates for horseradish peroxidase. It yields an intense brown product that is insoluble in both water and alcohol. It can be made more sensitive by adding metal salts such as cobalt or nickel to the substrate solution. - [Read Antibody Addition to Drosophila Specimens and Detection Using Enzyme-Linked Reagents Protocol]

Category: Drosophila Protocols

Protocol for antibody addition to Drosophila specimens and detection using fluorochrome-linked reagents. Fluorochrome-linked reagents should be used when high resolution is needed or if two antigens need to be localized simultaneously. Because of the thickness of fly specimens, detection requires access to a confocal microscope. - [Read Antibody Addition to Drosophila Specimens and Detection Using Fluorochrome-Linked Reagents Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

This method, for the selective amplification of full-length cDNA ends, involves the addition of an adapter during reverse transcription.  This method takes advantage of the propensity of Moloney murine leukemia virus reverse transcriptase (MMLV RT) to append two to four cytosines to the 3'-end of newly synthesized cDNA strands.  The additional residues are added when the enzyme reaches the 5'-cap structure at the end of the mRNA template. - [Read Cap-Switching RACE Protocol]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol described here produces chromatin with regularly spaced nucleosomes having physiological nucleosome repeat lengths; something that can be difficult to achieve with purified components. In addition, chromatin assembled with the S-190 Chromatin Assembly Extract contains the ATP-dependent chromatin remodeling factors necessary for efficient transcription. - [Read Chromatin Assembly on Template DNA with Transcription Factors and Drosophila S-190 Chromatin Assembl]

Category: PCR Protocols: PCR Cloning Protocols

Pairs of oligonucleotide primers used in PCR are often designed with restriction sites in their 5' regions.  In many cases, the sites are different in the two primers.  In this case, amplification generates a target fragment whose termini now carry new restriction sites that can be used for directional cloning into plasmid vectors.  The purified fragment and the vector are digested with the appropriate restriction enzymes, ligated together, and transformed into E. coli. - [Read Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA Protocol]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Culture conditions have been established for a second blastocyst-derived cell line, trophoblast stem (TS) cells, in addition to embryonic stem (ES) cells. This protocol describes a method for culturing TS cell lines. These cells can then be used to study trophoblast differentiation and placental function. - [Read Culturing Trophoblast Stem (TS) Cell Lines Protocol]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes how subcellular-sized particles are accelerated to high velocity to carry double-stranded RNA (dsRNA) into Drosophila embryos.  The major advantage of this procedure over microinjection (Microinjection of dsRNA into Drosophila Embryos) is that particle bombardment is easier and faster to perform.  In addition, the mechanical trauma received is far less than by microinjection, allowing better survival of embryos and fewer phenotypic artifacts. - [Read Delivery of dsRNA into Drosophila Embryos by Gene Gun Protocol]

Category: Signalling Signal Transduction Protocols

This kinase assay is meant to determine whether an agonist or event can influence the autophosphorylation of FAK. The addition of 1 μl of polyGT to the kinase reaction mix will determine the activity of the enzyme against a substrate. Includes information on: Harvest, Immunoprecipitation, Kinase Reaction and Antibody Detection of FAK. - [Read FAK Autophosphorylation Assay]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Flow cytometric determination of leukocyte surface antigens in whole blood. Quantitation of cell surface antigens in whole blood with the flow cytometer is very simple and requires:
1. Blood collection; 2. Addition of antibody; 3. Calibration of the flow cytometer; 4. Making measurements. - [Read Flow cytometric determination of leukocyte surface antigens in whole blood]

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for pulsed-flow microinjection using the Eppendorf FemtoJet injector and Eppendorf InjectMan; this is the most common type of pulsed-flow microinjection system currently being used. The advantage of this type of system over a controlled-flow system is that much more control is available over the injection parameters, reducing variability in injections. In addition, the system allows a diagonal insertion of the needle into the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System 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: Molecular Biology Protocols: Carbohydrate Protocols

Indirect method measuring immunofluorescence coupled to second antibody. Best for membrane antigens in addition to intra- and extracellular antigens, may be applied to frozen tissue sections, to cells in suspension, and to cells attached to glass slides or coverslips. Tadashi Tai~Head, Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan - [Read Immunohistochemistry using Anti-Ganglioside Antibodies]

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: Microbiology: Bacterial E.Coli Culture Media & Plates

LB Plates Pouring and Preparation. antibiotics addition. Janet Smith, Boston Biomedical Research Institute - [Read LB Plates Pouring and Preparation]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Describes the steps in detail to isolate and expand neural stem cells in the form of neurospheres from tissue dissections of the post-natal mouse brain. Procedures for the long term passage of neurospheres and the cryopreservation of neurospheres are also provided. In addition to the guidelines and tips for generating neurosphere cultures, we describe the method to prepare neurospheres for analysis by light microscopy. - [Read Neural Stem Cell Culture: Neurosphere Generation, Microscopical Analysis and Cryopreservation]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Plasmodium vivax is the second most prevalent malaria parasite affecting more than 75 million people each year, mostly in South America and Asia. In addition to major morbidity this parasite is associated with relapses and a reduction in birthweight. Practical PCR genotyping protocols based on polymorphic loci present in two P. vivax genetic markers. - [Read Practical PCR Genotyping Protocols for Plasmodium vivax using Pvcs and Pvmsp1]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. - [Read Preparation of Cells and Reagents for Flow Cytometry Protocols]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

siRNAs produced upon the addition of dsRNA to Drosophila embryo extract are enriched in a micrococcus-nuclease-resistant fraction.  After proteinase K treatment and dephosphorylation with calf intestinal phosphatase, these siRNAs mediate efficient RNAi in vitro. - [Read Preparation of siRNAs from Drosophila Embryo Extracts Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

This protocol is not phenol-based, but does require the addition of chloroform. The Concert Plant Reagent is intended for the isolation of RNA from a wide variety of plant tissues including blue spruce needles, potato tuber etc. - [Read Purification of RNA from Plant Tissue Using the Concert Plant Reagent]

Category: Immunology: T Cell Protocols

In the first protocol, IL-2-producing murine T cells are measured following stimulation by the mitogen Con A. The second protocol provides a modification for using human responder cells. The second protocol is used for estimating the proportion of cells that can generate a clone of cytotoxic effector cells when stimulated by Con A with the addition of IL-2. - [Read Quantitation of Functional T Cells by Limiting Dilution Protocols]