Protocol for 96-well confirmation Yeast PCR. Includes: Clonal purification; Generate a master plate (96-well format); Making a frozen backup stock; Confirmation PCR for one Row; ORF Specific Confirmation PCR --> "A-B" primers (upstream junction); Transfer template DNA to multiwell PCR plate; Prepare and dispense master mix for A-B PCR. - [Read 96-Well Confirmation Yeast PCR Protocol]
This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, in cultured adherent RAW 264.7 cells, using a 96-well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fluo-3, which permeates cell membranes as an ester and is
hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells]
This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, in cultured adherent RAW 264.7 cells, using a 96- well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fluo-3, which permeates cell membranes as an ester and is
hydrolyzed in the cell to its Ca2+-sensitive acidic form. Fluorescence for the adherent cells is measured over time by using a bottom read of a 96-well plate, with cells that have been washed. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fluo-3 Protocol]
This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+], in cultured adherent RAW 264.7 cells, using a 96-well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fura-2, which permeates cell membranes as an ester and is
hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fura-2 (with FLEXstation)]
This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i , in cultured adherent RAW 264.7 cells, using a 96- well plate format. This objective is accomplished by using the Ca2+-sensitive fluorescent dye, fura-2, which permeates cell membranes as an ester and is
hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fura-2 Protocol]
4 strains of E. coli are used in these studies: JM101 for M13 infection and isolation, XL1BMRF'for M13 or pUC-based DNA transformation, and ED8767 for cosmid DNA transformation. To maintain their respective F' episomes necessary for M13 viral infection, JM101 is streaked onto a M9 minimal media plate and XL1BMRF' is streaked onto an LB plate containing tetracycline. ED8767 is streaked onto an LB plate. These plates are incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid.. - [Read Bacterial Cell Maintenance Protocol]
Protocol for cloning genes from a phage library. Includes: Titer and plate out phage; Lift plaques onto filters and prepare them for screening; Make a probe; Hybridize the probe to the filters; Wash the filters and expose to film; Purify putative plaques; Excise plasmid from the desired phage. - [Read Clone Genes From a Phage Library Protocol]
Flow assays offer visualization of cell adhesion under wall shear stress. Visualization of the different events of cell adhesion can be quantified by selective image acquisition and subsequent image processing. Flow assays are suited for adhesive events which occur very rapidly in a time scale shorter than that of most static adhesion assays. Also, events subsequent to the initial events can be studied such as cell stabilization and spreading giving some insight into the kinetics of cell-cell. - [Read Dynamic Flow Assay for Cell Adhesion in a Parallel Plate Flow Chamber]
Flow assays allow visualization of cell adhesion under well-defined wall shear stress. Visualization of the events of cell adhesion are quantified by selective image acquisition and image processing. Events subsequent to the initial events can be studied such as cell stabilization and spreading. John T. Patton~GlycoTech Corporation, Rockville, Maryland - [Read Dynamic Flow Assay in a Parallel Plate Flow Chamber]
Protocol for the electroporation of ES cells. Cells are routinely passaged two days prior to electroporating. Usually one 10 cm plate at approximately 80% confluency will provide enough cells for 1-2 electroporations. - [Read Electroporation of ES Cells Protocol]
Protocol for ELISA assay for NGF. Includes: ABSORPTION OF THE POLYCLONAL AND PREIMMUNE SERUM; BLOCKING; SAMPLE PREPARATION; PREPARATION OF NGF STANDARDS; PROTEIN RECOVERY; DESIGNING THE PLATE; APPLYING THE STANDARDS AND SAMPLES; APPLYING THE MONOCLONAL; APPLYING SECONDARY ANTIBODY; APPLYING STREPTAVIDIN; CHROMAGEN DEVELOPMENT; READING THE PLATE. - [Read Enzyme-Linked ImmunoSorbent Assay (ELISA) for NGF Protocol]
Most manipulations with M13, including preparations of viral stocks and isolation of single- and double-stranded DNAs, begin with small-scale liquid cultures that are infected with an M13 plaque, picked from an agar plate. - [Read Growing Bacteriophage M13 in Liquid Culture Protocol]
The transfer of proteins from a Tris/glycine SDS-polyacrylamide gel to a membrane using a semi-dry method is achieved by placing the gel next to a piece of nitrocellulose filter. This sandwich is placed directly between two plate electrodes, and the proteins are then transferred from the gel onto the filter. - [Read Immunoblotting: Semi-Dry Electrophoretic Transfer of Proteins from Gels to Membranes Protocol]
Protocol describes a plate method that gives very good yield for cloning. Includes: T-TYN Media + Mg+2; T-TYN Plates; T-TYN Top Agarose. - [Read Lambda DNA Preparation Protocol]
Protocol for large-scale yeast transformation. Includes: Yeast Cell Preparation; Large Scale Transformation; To plate on solid medium; To select in liquid. - [Read Large-Scale Yeast Transformation Protocol]
Assay measures cell viability. It is a two-color fluorescence assay that simultaneously determines Live cell number and Dead cell number. This protocol is designed for use with the GEMINI XS Microplate Spectrofluorometer, a multi-well plate scanner with dual excitation/emission capabilities, but the assay is also adaptable for flow cytometry and fluorescence microscopy. Includes: Cell Culture; Preparation for the Assay; Live/Dead Assay; Reading the Plate; Data Analysis; Alternative protocol. - [Read Live/Dead Assay for Cell Viability Protoco]
Yeast strains may be stored indefinitely at low temperatures (-80 degrees C). Two archiving methods are presented below. In Method A , the cells are grown on a plate, while in Method B the cells are grown in liquid culture. - [Read Long Term Storage of Yeast Stocks Protocol]
Luciferase assay using a 24 well plate. Includes: Cell Lysis; Reagent Preparation; Luminescence measurement; Wash injector; Protein quantitation; Data Analysis. - [Read Luciferase Assay 24 Well Plate (Promega System)]
A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.
A protocol for the selection of Phage Antibodies using Immobilized Antigen. This method describes the selection of antibodies from bacteriophage antibody libraries that recognize a specific antigen. The phage display library of antibody-displaying phage particles is exposed to antigen attached to a solid substrate (Nunc Immuno™ tubes). The phage particles with affinity for antigen bind to the immobilized antigen and are selected from the library of phage expressing antibodies.
Unlike spherical phage, such as T4 and λ, which have roughly equal weight ratios of protein to DNA, filamentous phage have about six times more protein than DNA; the protein therefore contributes substantially to the absorption spectrum.
3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol. This protocol contains the steps for 3' end rapid amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly-A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.
This protocol a protocol on how to generate transfected embryonic stem (ES) cell clones. The previous protocol in this series is the Protocol for Electroporation of ES cells. The next protocol in the series is the Protocol on Disaggregation, Expansion, and Freezing of Transfected ES Clones.
This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.
The protocol gives general considerations for the design of targeting vectors for transgenic mice. The protocol shares tips in the design of knock-out and knock-in vectors and some of their strategies for producing homologously recombined embryonic stem cells.