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ELISPOT Protocol Detailed PDF - http://www.biolegend.com/images/Protocols/BioLegend%20ELISPOT%20protocol.pdf

Category: ELISA Protocols: ELISpot Protocols

Coat the Plate, Block the Plate, Add Detection Antibody, Solutions and buffers for ELISpot. Biolegend. - [Read ELISPOT Protocol Detailed PDF]

M9 Plate Supplementation Table - http://www.fhcrc.org/science/labs/gottschling/bac/bsup.html

Category: Microbiology: Bacterial E.Coli Culture Media & Plates

M9 Plate Supplementation Table. URA, LEU, MET, HIS, TRP. Fred Hutchinson Cancer Research Center - [Read M9 Plate Supplementation Table]

Mouse Embryonic Fibroblast Culture and Propagation Part II PDF - http://www.wicell.org/uploads/media/Protocols_Part_2_05.pdf

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: MEF Protocols Mouse Embryonic Fibroblasts

WiCell Reserarch Institute. Count MEF Cells, Trypsinize, Split, Freeze, Thaw, Irradiate, Reconstitute, Plate, Feeder. - [Read Mouse Embryonic Fibroblast Culture and Propagation Part II PDF]

Protocol for Luciferase Assay for In Vitro Detection - http://aesop.rutgers.edu/~lamlab/lucassayprotocol.html

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol for luciferase assay for in vitro detection. Protocol includes: Before cell lysate preparation; Cell lysate preparation; Protocol for manual luminometers; Protocol for plate reading luminometer; Preparation of protein assay reagent; Protein standards. - [Read Protocol for Luciferase Assay for In Vitro Detection]

Luciferase Assay 24 Well Plate (Promega System) - http://www.biochem.northwestern.edu/ibis/morimoto/research/Protocols/II.%20Eukaryotes/F.%20Luciferase%20Assays/2a.single%20luciferase.pdf

Category: Reporter Gene Assays: Luciferase Assay Protocols

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)]

Removal of Yeast Contamination from Lymphoblast Cultures Protocol - http://humgen.wustl.edu/hdk_lab_manual/hcc/hcc8.html

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

This method is advantageous for saving the occasional cultures that become contaminated. Yeast contaminated cultures will appear cloudy when slightly shaken and lymphocytes will not cluster together as much as normal. If cultures are suspect, a drop of culture can be streaked on a YPD media plate to check for growth of yeast colonies, or a 5 ml sample can be taken to Barnes Diagnostic Center for identification of yeast strain. - [Read Removal of Yeast Contamination from Lymphoblast Cultures Protocol]

Assay of Intracellular Free Calcium in RAW 264.7 Cells - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000210.pdf?pid=PP00000210

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

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]

Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fura-2 (with FLEXstation) - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000211.pdf?pid=PP00000211

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

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)]

Bacterial Cell Maintenance Protocol - http://www.genome.ou.edu/protocol_book/protocol_partI.html#I.G

Category: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

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]

Long Term Storage of Yeast Stocks Protocol - http://humgen.wustl.edu/hdk_lab_manual/yeast/yeast2.html

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

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]

Measurement of Cell Adhesion Under Static Conditions - http://www.glycotech.com/protocols/Proto8.html

Category: Cell Biology and Cell Culture: Cell Adhesion Protocols

Many proteins and molecules promote cell adhesion including several cell surface carbohydrate binding proteins. Cell adhesion measurements on 96-well microtiter plate format are difficult due to the shear forces generated by washing the wells. The protocol here introduces the use of a liquid-filled wash chamber that separates unbound cells by gravity. This eliminates uncontrolled shear forces and passage of adherent cells through a liquid/air interface. John L. Magnani~GlycoTech Corporation. - [Read Measurement of Cell Adhesion Under Static Conditions]

MTT Assay for Detection of Cellular Proliferation - http://www.ebioscience.com/ebioscience/appls/AC145.htm#mouse

Category: Immunology: T Cell Protocols

Mouse Protocol: Stimulation of mouse peripheral T cells with plate-bound 145-2C11 monoclonal antibody; MTT assay for detection of cellular proliferation. - [Read MTT Assay for Detection of Cellular Proliferation]

Live/Dead Assay for Cell Viability Protoco - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000023.pdf?pid=PP00000023

Category: Cell Biology and Cell Culture: Cell Viability Protocols

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]

Elispot plates column - http://www.zellnet.com/elispotplatescolumn/

Category: ELISA Protocols: ELISpot Protocols

Tips for selecting the â€œrightâ€ ELISPOT filter plate and making the best protocol choices. Alan J. Weiss ZellNet - [Read Elispot plates column]

Preparation of Seeded NGM Plates For Worm Food Protocol - http://www.bio.com/protocolstools/protocol.jhtml?id=p1558

Category: C. elegans Protocols: C. elegans Cell Culture Protocols

Protocol describes how to seed plates with E. coli OP50 for C. elegans cultures. Includes: Bacterial Stock Plate Preparation; Bacterial Broth Preparation; Seeding Bacteria Plates for Culturing Worms. - [Read Preparation of Seeded NGM Plates For Worm Food Protocol]

YC Dropout Plate Supplementation Table - http://www.fhcrc.org/science/labs/gottschling/yeast/ycsup.html

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

YC Dropout Plate Supplementation Table. - [Read YC Dropout Plate Supplementation Table]

Streaking Yeast Stocks Protocols - http://humgen.wustl.edu/hdk_lab_manual/yeast/yeast1.html

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

Some yeast strains are unstable (e. g., small YAC-bearing strains) and need to be repurified by streaking on an agar plate and then verifying the genetic content of the isolated colony before proceeding. In cases where the strain is unstable, plan to streak the cells onto the selective medium to retain the desired stock, (however, most strains can be streaked onto the complete medium, YPD). - [Read Streaking Yeast Stocks Protocols]

Immunoblotting: Semi-Dry Electrophoretic Transfer of Proteins from Gels to Membranes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4301

Category: Western Blot Protocols: General Western Blot Methods Protocols

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]

Lambda plate lysis Protocol - http://www.rpgroup.caltech.edu/~paul/protocols/phage/lambda/plate/

Category: Molecular Biology Protocols: Phage Protocols and Methods: Lambda Phage Protocols

Phage Protocols - Lambda plate lysis. Rob Philip Group. - [Read Lambda plate lysis Protocol]

Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fura-2 Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000211.pdf?pid=PP00000211

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

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]

Dynamic Flow Assay in a Parallel Plate Flow Chamber - http://www.glycotech.com/protocols/proto7.html

Category: Cell Biology and Cell Culture: Cell Adhesion Protocols

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]

Assay of Intracellular Free Calcium in RAW 264.7 Cells Loaded with Fluo-3 Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000210.pdf?pid=PP00000210

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

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]

Large-Scale Yeast Transformation Protocol - http://ygac.med.yale.edu/mtn/LS_transf_prot.stm

Category: Yeast Protocols: Yeast Transformation Protocols

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]

Enzyme-Linked ImmunoSorbent Assay (ELISA) for NGF Protocol - http://crutcherlab.med.uc.edu/ELI.htm

Category: Immunology: Immunoassays

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]

96-Well Confirmation Yeast PCR Protocol - http://sequence-www.stanford.edu/group/yeast_deletion_project/96_well_confirmation.html

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

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]

Articles (1-9 of 9)

Single Stranded Plasmid DNA Isolation Protocol

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.

Antibodies Phage Expression Protocol Using 96-well Microtiter Plates

A Phage display expression protocol for antibody expression in 96-well microtiter plates.

Immobilized Antigen Phage Antibody Selection Protocol

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.

Coupling Cysteine Containing Peptides to Carrier Protein for Immunization and Polyclonal Antibody

This protocol is used in the production of polyclonal antibodies that recognize a peptide sequence of interest.

Absorption Spectroscopy and Quantification of Filamentous Phage Protocol

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

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.

Picking Transfected ES Cell Clones Protocol

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.

Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

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.

Vector Design Protocol for Transgenic Mice Generation

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.

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