requires Protocols

Category: Yeast Protocols: Yeast Genetics Protocols

Protocol describes an assay where it requires growing saturated cultures of yeast, counting, and spotting serial dilutions of yeast on both CSM and CSM + 6AU plates. - [Read 6-Azauracil Sensitivity Assay for Yeast Protocol]

Category: PCR Protocols: Bisulfite Conversion Based PCR Protocols

Protocol for the analysis of DNA methylation using bisulphite sequencing. Method allows precise analysis of methylation in a certain region by converting all nonmethylated cytosines into tymines, while methylated cytosines remain unchanged.  This method requires small amount of genomic DNA and therefore seems to be very useful for the analysis of clinical samples, where the material amount is limited.
- [Read Analysis of DNA Methylation using Bisulphite Sequencing 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: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for producing diploid embryo-tetraploid embryo chimeras. It requires the timed combination of four-cell-stage tetraploid embryo production and the procedure for diploid embryo-diploid embryo aggregation. The resulting chimeras are useful for phenotypic analysis when an induced mutation has an extraembryonic phenotype. - [Read Assembling Aggregates between Diploid and Tetraploid Embryos Protocol]

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for producing ES cell-tetraploid embryo chimeras. It requires the timed combination of four-cell-stage tetraploid embryo production and the procedure for ES cell-diploid embryo aggregation in which diploid embryos are replaced with tetraploid embryos. The resulting chimeras can be used to analyze the embryonic versus extraembryonic phenotype of a mutation. - [Read Assembling Aggregates between Embryonic Stem (ES) Cells and Tetraploid Embryos Protocol]

Category: Cell Culture Protocols: Sterile Technique Protocols

Cultured mammalian cells are used extensively in cell biology studies; it requires a number of special skills in order to be able to preserve the structure, function, behavior and biology of the cells. This unit describes the basic skills required to maintain and preserve cell cultures: aseptic technique, medium characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. - [Read Basic Techniques for Mammalian Cell Tissue Culture Protocol]

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

Cultured mammalian cells are used extensively in cell biology studies; it requires a number of special skills in order to be able to preserve the structure, function, behavior and biology of the cells. This unit describes the basic skills required to maintain and preserve cell cultures: aseptic technique, medium characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. - [Read Basic Techniques for Mammalian Cell Tissue Culture Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Manual measurement and manipulation of the cell surface requires access to the cells, usually in an open chamber. Temperature-controlled chambers or stage inserts are preferred for maintaining physiological activity during the experiment. For example, heated culture dishes with coverslip glass bottoms (Bioptechs) permit high-resolution fluorescence microscopy of living cells during force application. - [Read Chambers for Examination of Live Cells under Mechanical Stress Protocol]

Category: Nucleic Acid Purification Protocols

Ultrafiltration is an alternative to ethanol precipitation for the concentration and desalting of nucleic acid solutions.  It requires no phase change and is particularly useful for dealing with very low concentrations of nucleic acids.  This protocol describes the use of the Microcon cartridge, a centrifugal ultrafiltration device, to concentrate and desalt nucleic acid solutions. - [Read Concentrating and Desalting Nucleic Acids with Microconcentrators Protocol]

Category: Microscopy Protocols: Confocal Microscopy Protocols

There are two major forms of laser scanning microscopy: confocal laser scanning microscopy (CLSM) and multiphoton laser scanning microscopy (MPLSM). Information on: X-t scans and X-Y scans; Confocal Laser Scanning Microscopy; Multiphoton Laser Scanning Microscopy; MPLSM requires no pin hole; Advantages of MPLSM over CLSM. - [Read Confocal Laser Scanning Microscopy]

Category: Cytogenetics Protocols

CGH provides a comprehensive picture of all chromosomal gains and losses within a single experiment. In contrast to banding analysis, CGH requires no preparation of metaphase chromosomes from the cells to be analyzed (which in certain tumor types may be difficult or even impossible). - [Read Detection of Chromosomal Imbalances Using DOP-PCR and Comparative Genomic Hybridization (CGH)]

Category: Cloning Protocols: Vector Protocols

Directional cloning requires that the plasmid vector be cleaved with two restriction enzymes that generate incompatible termini and that the fragment of DNA to be cloned carries termini that are compatible with those of the doubly cleaved vector. - [Read Directional Cloning into Plasmid Vectors Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

The original maize DNA miniprep protocol is used extensively for many plant species and different tissues. This slightly modified version is acceptable for most DNA extractions. The procedure has the advantage of isolating DNA from plant material very rapidly. The procedure requires a table-top drill-press (mechanized homogenizer). - [Read DNA Microprep Isolation from Plants Protocol]

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors.  This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. - [Read ELISA Method Measure Inhibition COX Enzymes]

Category: C. elegans Protocols: C. elegans Fixing Protocols

Common method for fixing worms is to use paraformaldehyde. This method provides a gentler fixation than the Bouin’s method, but often requires the use of collagenase. This method is particularly good for examining adult worms. - [Read Fixing Caenorhabditis elegans in Paraformaldehyde Protocol]

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: E Coli Protocols: E coli Genetics Protocols

Protocol for the generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system. Technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. - [Read Generation of Gene Deletions and Gene Replacements in Escherichia coli 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: Cloning Protocols

Protocol for ligating plasmid and target DNAs in low-melting-temperature agarose. Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs. - [Read Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Protocol]

Category: Chromatography Protocols: Gas Chromatography Protocols

Linkage analysis provides information on sugar type, ring size, and substitution positions for each monosaccharide.  The method in this protocol, using NaOH as the base, is one of the simpler linkage analysis methods.  It requires approximately 1-5 µg of carbohydrate. - [Read Linkage Analysis Using the NaOH Methylation Method Protocol]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol details the preparation of biotin-labeled target samples and hybridization of these samples to an Affymetrix in situ synthesized oligonucleotide GeneChip array.  The procedure requires a minimum of 5 µg of purified total RNA as starting material. - [Read Microarray Protocol for Affymetrix In Situ Synthesized Oligo Arrays]

Category: Microarray Protocols: Amino-allyl Microarray Methods

Protocol details the preparation of fluorescently labeled target samples (aminoallyl method) and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides. The procedure requires a minimum of 3 µg of purified total RNA as starting material. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized]

Category: Microarray Protocols: Microarray DNA Labeling Protocols

Protocol details the preparation of fluorescently labeled target samples and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides. The procedure requires a minimum of 3 µg of purified total RNA as starting material. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized Oligo Array]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol details the preparation of fluorescently labeled target samples and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides.  The procedure requires a minimum of 3 µg of purified total RNA as starting material. Includes: cDNA Synthesis; Fluorescent cRNA Synthesis; cRNA Precipitation and Cleanup; cRNA Quantification; Hybridization; Washing. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized Oligo Arrays]

Category: Microscopy Protocols: Optical Microscopy Protocols

Diffraction-limited optical microscopy requires that the spatial resolution of an image is limited by the wavelength of the incident light & by the numerical apertures of the condenser & objective lens systems.The development of near-field scanning optical microscopy (scanning near-field optical microscopy) has allowed for a imaging technique that retains the various contrast mechanisms afforded by optical microscopy methods while attaining spatial resolution beyond the optical diffraction limit - [Read Near-Field Scanning Optical Microscopy]