procedures Protocols

Category: DNA Microarray Protocols

Hegde et al., Array Fabrication, microarray Printing, Probe Preparation and Hybridization, RNA Extraction, RNA labeling, Data Collection, Normalization, and Analysis.Institute for Genomic Research, Rockville, MD - [Read A Concise Guide to cArray Hybridization Procedures - DNA Microarray Analysis]

Category: Cell Culture Protocols: Sterile Technique Protocols

This unit describes some of the ways that a laboratory can deal with the constant threat of microbial contamination in cell cultures. A protocol on aseptic technique is described first. This catch-all term universally appears in any set of instructions pertaining to procedures in which noncontaminating conditions must be maintained. - [Read Aseptic Technique for Cell Culture Protocol]

Category: RNA Protocols: cDNA Libraries Library

Includes procedures for cDNA screens using secondary and tertiary screening. Dr. Bart Frank Lab. - [Read Benton Davis Blots for cDNA Libraries]

Category: Cell Biology and Cell Culture

OptiPrep Application sheet C14 describes procedures for determining the density and sedimenting properties of any cell (of any size or density) using either a continuous or discontinuous gradient of iodixanol. This Application Sheet describes procedures aimed at isolating specifically a relatively low-density cell fraction from any tissue. - [Read C25 Isolation from spleen, thymus, pancreas, alveolar tissue and other tissues]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Sorting Protocols

The combination of prospective identification/isolation of bone marrow progenitors and quantitative RT-PCR is a powerful tool to understand the molecular mechanism underlying hematopoiesis. Describes the standard procedures of the murine myeloid progenitor staining for fluorescence activated cells sorting (FACS) and RNA purification methods. - [Read Cell Staining for Sorting of Hematopoietic Stem Cells (HSC) and Myeloid Progenitors]

Category: Stem Cell Protocols

The combination of prospective identification/isolation of bone marrow progenitors and quantitative RT-PCR is a powerful tool to understand the molecular mechanism underlying hematopoiesis. Here, we described our standard procedures of the murine myeloid progenitor staining for fluorescence activated cells sorting (FACS) and RNA purification methods. - [Read Cell Staining for Sorting of Hematopoietic Stem Cells and Myeloid Progenitors and Isolating RNA]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Immobilized metal ion affinity chromatography (IMAC) exploits a molecule’s affinity for chelated metal ions.  The amino acid histidine present in many proteins forms complexes with transition metal ions such as Cu2+, Zn2+, Ni2+ and Fe3+.  Chelating Sepharose™ Fast Flow with a suitable immobilized metal ion will therefore selectively retain proteins with exposed histidine. - [Read Chelating Sepharose Fast Flow Protocol]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Cloning Enzymes a Guide Promega. An Enzyme Resource Guide series, highlights those enzymes important in nucleic acid cloning procedures. Promega - [Read Cloning Enzymes a Guide Promega]

Category: Reporter Gene Assays: GFP Assay Protocols

The following protocol can be used for the development of stable cell lines expressing GFP fusion proteins. Although optimal transfection procedures (e.g., calcium phosphate, electroporation, or FuGENE 6 [Roche Applied Science]) vary depending on cell type, this general transfection procedure has been successful for stable transfection of HeLa, A-431, U2OS, BHK, and HT1080 cells. - [Read Constructing and Expressing GFP Fusion Proteins]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

The starting material for de novo isolation of stem cell lines can be either normal 3.5-days post coitum (dpc) expanded blastocysts or "delayed" blastocysts. Delayed blastocysts are usually collected 4-6 days after ovariectomy. For both groups of blastocysts, tissue culture procedures are similar. The only difference is the timing of the first disaggregation, because delayed blastocysts will initially grow more slowly. - [Read De Novo Isolation of Embryonic Stem (ES) Cell Lines from Blastocysts Protocol]

Category: RNA Protocols: cDNA Libraries Library

Differential cDNA Screening Procedures. The protocols listed refer to cDNA library construction and preliminary differential screening procedures. Nick Talbot lab - [Read Differential cDNA Screening Procedures]

Category: DNA Protocols: DNA Cloning

Agarose gel purification, Annealing and extending, oligonucleotides, Ethanol Precipitation, Ligation Miniprep, Oligonucleotide purification, Recovering DNA bands, Restriction digest Gene Clean. The Hu Lab. - [Read DNA cloning Procedures]

Category: DNA Microarray Protocols

Protocols, Frequently Asked Questions, Validation. Dr. Bart Frank - [Read DNA Microarray Procedures]

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: Microscopy Protocols: Electron Microscopy Protocols

Article describe the preparation of cells for correlative electron microscopy after live light microscopic observation of fluorescently labeled cytoskeletal proteins microinjected into the same cells. Since identification of cytoskeletal elements in electron microscopic preparations is an essential part of any correlative study, procedures for immunogold labeling of cytoskeletal components and for myosin S1 decoration of actin filaments are also described. - [Read Electron Microscopy of the Cytoskeleton of Cultured Cells]

Category: PCR Protocols: PCR Optimization

Method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield.  The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. - [Read Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization Protocol]

Category: Protein Microarray Protocols

Experimental Procedures for Protein Microarrays. MacBeath G & Schreiber SL (2000). Printing proteins as microarrays for high-throughput function determination. Science 289: 1760-1763. - [Read Experimental Procedures for Protein Microarrays]

Category: Developmental Biology: Utilizing Model Organisms Protocols

In the experiment there are three players A, B, and C. Player A gets an initial endowment of 10, player B gets 0 and player C gets 5 points. First, player A decides how many of his 10 points to transfer to B. Then, player C observes the decisions of A and gets the possibility to leave the payoffs unaffected or to punish A by deducting 3 (or 6) points from A at a cost of 1 point (or 2 points) for C.... - [Read Experiments on Parochial Altruism in Humans; Procedures and Instructions]

Category: Recombinant Protein Protocols

Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography. Biological Procedures Online - [Read Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affin]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: TUNEL assay apoptosis

FACS Procedures for Apoptosis Detection with TUNEL. U. Pennsylvania Dental School. - [Read FACS Procedures for Apoptosis Detection TUNEL]

Category: Histology Protocols: Fixation of Cells Tissues Protocol

Fixation is to preserve cells and tissue constituents in as close a life-like state as possible and to allow them to undergo further preparative procedures without change. Fixation arrests autolysis and bacterial decomposition and stabilises the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing. Great detailed guide with protocols. Anthony S-Y Leong. - [Read Fixation and Fixatives - A great guide]

Category: Molecular Imaging: Immunofluorescence Microscopy

Fluorescent Staining of Cells protocol. Yu-li Wang Lab. Fluorescent Phalloidin Staining, Immunofluorescence Staining and procedures for staining. - [Read Fluorescent Staining of Cells]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

The procedures involve the isolation and growth of primary cell cultures from rodent and human tissue as well as the use of viral vectors for the introduction and expression of mammalian genes in cells in culture and in live rodents. - [Read Growth of Primary Cell Culture and Viral Vector Handling Protocols]

Category: Immunohistochemistry Protocols: Antibody Staining Protocols

There are several strategies to visualize the antibody. For transmitted light microscopy, color development substrates for enzymes are often used. The antibody can be directly labeled with the enzyme. However, such a covalent link between an antibody and an enzyme might result in a loss of both enzyme and antibody activity. For these reasons several multistep staining procedures have been developed, where intermediate link antibodies are used. In this protocol use the Vectastain ABC-kit. - [Read Immunocytochemistry in Free-Floating Sections Protocol]

Category: Immunology: Immunofluorescence Protocols

Protocol for immunofluorescence labeling of cells. Includes: Cell Preparation; Fixation; Application of Primary Antibody; Application of Secondary Antibody and Evaulation. - [Read Immunofluorescence Labeling of Cells Protocol]