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CHO Centrosome Prep Protocol - http://mitchison.med.harvard.edu/protocols/mt1.html

Category: Cell Organelle Protocols: Nucleus Protocols

The key step is the lysis which solubilizes centrosomes away from nuclei by very low ionic strength lysis after treatment of cells with nocodazole and cytochalasin B. The released centrosomes are then centrifuged onto a Ficoll cushion (to avoid pelleting) and the interface between the lysate and the Ficoll is collected and the centrosomes are concentrated on a sucrose gradient. Fractions are assayed by spindown and double IF with 5051 serum and anti-tubulin and the pooled fractions are frozen... - [Read CHO Centrosome Prep Protocol]

Chromatin Immunoprecipitation (ChIP) of Protein Complexes Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4560

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Protocol describes the use of chromatin immunoprecipitation technology (ChIP) to analyze interactions of proteins or protein complexes with DNA in vivo. In this approach, the material is fixed with formaldehyde to preserve DNA-protein and protein-protein associations, the cells are lysed, and the chromatin is cut and solubilized. The chromatin suspension is immunoprecipitated with an antibody against the protein(s) of interest, and the coimmunoprecipitated DNA fragments are analyzed. - [Read Chromatin Immunoprecipitation (ChIP) of Protein Complexes Protocol]

CHROMATIN IMMUNOPRECIPITATION (CHIP) PROTOCOL FOR YEAST - http://www.ciwemb.edu/labs/koshland/Protocols/YEAST/chipmod.html

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

SPHEROPLAST CELLS, WASH SPHEROPLASTS, SONICATE SPHEROPLASTS, CHROMATIN SOLUTION, SET UP IMMUNOPRECIPITATIONS, REVERSE FORMALDEHYDE CROSSLINKS. Paul - Koshland lab. - [Read CHROMATIN IMMUNOPRECIPITATION (CHIP) PROTOCOL FOR YEAST]

Chromatin Immunoprecipitation (ChIPs) Protocol - http://genomecenter.ucdavis.edu/farnham/farnham/protocols/chips.html

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

IP Wash buffer recipes and protocol for ChIP Assay. Protocol works with NIH 3T3, Friend, HeLa, Raji and CHO cells.. Farnham Lab - [Read Chromatin Immunoprecipitation (ChIPs) Protocol]

Chromatin Immunoprecipitation and Microarray-Based Analysis of Protein Location Protocol - http://www.nature.com/nprot/journal/v1/n2/abs/nprot.2006.98.html

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. - [Read Chromatin Immunoprecipitation and Microarray-Based Analysis of Protein Location Protocol]

Chromatin IP (CHIP assay) - http://www.fhcrc.org/science/labs/breeden/Methods/chromatinIP.html

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

ChIP assay protocol with 2 steps: in vivo formaldehyde cross-linking of whole cells and protein-protein and protein-DNA interactions, followed by immunoprecipitation of protein-DNA complexes with specific antibodies from sonicated extracts. Breeden Lab - [Read Chromatin IP (CHIP assay)]

Chromosomal DNA Preparation Protocol - http://genetics.med.harvard.edu/~cepko/protocol/mike/C3.html

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Protocol for chromosomal DNA preparation. Protocol was developed for cultured cells but should be appropriate for dissociated tissues as well. - [Read Chromosomal DNA Preparation Protocol]

Collecting Blastocysts Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4360

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol describes a method for collecting blastocysts from pregnant female mice at 3.5 to 4.5 days post coitum (dpc). The blastocysts can then be injected with embryonic stem cells to make chimeras. - [Read Collecting Blastocysts Protocol]

Collecting Zygotes and Removing Cumulus Cells With Hyaluronidase Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4358

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol describes a method for collecting zygotes from pregnant female mice. The zygotes can then be used for microinjection. - [Read Collecting Zygotes and Removing Cumulus Cells With Hyaluronidase Protocol]

Collection of Mouse Peritoneal Cells Protocol - http://icg.cpmc.columbia.edu/cattoretti/Protocol/mousepathology/CollectPerC.html

Category: Mouse Protocols

Protocol for the collection of mouse peritoneal cells. - [Read Collection of Mouse Peritoneal Cells Protocol]

Colorimetric Cytotoxcity Assays for Anchorage Dependent Cells Protocol - http://embryo.ib.amwaw.edu.pl/invittox/prot/43.htm

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

The potential cytotoxicity of compounds under hypoxic conditions is determined by exposing cell cultures to test compounds in a low oxygen atmosphere. Subsequent cell survival is determined by the MTT and methylene blue colorimetric assays. - [Read Colorimetric Cytotoxcity Assays for Anchorage Dependent Cells Protocol]

Competent cell preparation - http://www.chem.uga.edu/scottgrp/GrpProtocols/Competent_cell_preparation.htm

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Bacterial E.Coli competent cell preparation. Calcium Chloride Protocol, Preparation of calcium chloride competent cells for frozen storage, Electroporation Protocol, Electroporation Protocol for transformations using double-stranded plasmids, Reference: - [Read Competent cell preparation]

Competent Cell Preparation [CaCl2] - http://www.cf.ac.uk/biosi/staff/kille/Methods/Gene/Comp_cell_CaCl.html

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Nice protocol for the preparation of Bacterial cells to allow uptake of plasmid vectors. Dr Peter Kille. Hot Metals Cardiff. - [Read Competent Cell Preparation [CaCl2]]

Competent cells, calcium chloride method, E. coli, description - http://web.uct.ac.za/depts/mmi/bbhelp/bac1.html

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Competent cells, calcium chloride method, E. coli, description. CaCl2 and DMSO methods for preparing bacterial and E.Coli competent cells. Zappe, H. BBHelp. - [Read Competent cells, calcium chloride method, E. coli, description]

Confocal Microscopy Specimen Preparation Using Synthetic Fluorophores and Immunofluorescence - http://www.olympusconfocal.com/applications/protocols/index.html

Category: Microscopy Protocols: Confocal Microscopy Protocols

Includes staining protocols: Triple-Staining Adherent Cells with MitoTracker, Phalloidin,& Nuclear Dyes; Staining Adherent Cells with Cytokeratin Primary Antibodies & Synthetic Fluorophores; Staining Adherent Cells with Intermediate Filament Primary Antibodies & Synthetic Fluorophores; Staining Cells & Tissue Cryosections with Tubulin Primary Antibodies, Phallotoxins,& Synthetic Fluorophores; Triple-Staining Tissue Cryosections with Wheat Germ Agglutinin, Phalloidin,& Nuclear Dyes; etc. - [Read Confocal Microscopy Specimen Preparation Using Synthetic Fluorophores and Immunofluorescence]

Constructing and Expressing GFP Fusion Proteins - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4649

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]

Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4342

Category: Reporter Gene Assays: Luciferase Assay Protocols

It is possible that some cell lines have lost the ability to perform RNAi or that cells derived from certain tissues do not support RNAi. This reporter assay, for RNAi in mammalian cells, can be used to establish whether the cells under study are susceptible to RNAi. - [Read Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol]

Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4342

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

This reporter assay, for RNAi in mammalian cells, can be used to establish whether the cells under study are susceptible to RNAi. - [Read Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol]

Cryogenic Preservation and Storage of Animal Cells - Corning Life Science. PDF. - http://www.corning.com/Lifesciences/technical_information/techDocs/Cell_freezing_protocol.pdf

Category: Cell Biology and Cell Culture: Cell Culture Techniques: Cell Line Preservation Freezing

Cryogenic Preservation and Storage of Animal Cells. PDF.Corning Life Science. Very nice, detailed procedure for the freezing and storage of various cell lines. - [Read Cryogenic Preservation and Storage of Animal Cells - Corning Life Science. PDF.]

Cryogenic Preservation and Storage of Animal Cells Protocol - http://www.corning.com/Lifesciences/technical_information/techDocs/Cell_freezing_protocol.pdf

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

Cryogenic preservation (storage below -100°C) of cell cultures is widely used to maintain backups or reserves of cells without the associated effort and expense of feeding and caring for them. The success of the freezing process depends on four critical areas: Proper handling and gentle harvesting of the cultures; Correct use of the cryoprotective agent; A controlled rate of freezing; Storage under proper cryogenic conditions. - [Read Cryogenic Preservation and Storage of Animal Cells Protocol]

Cryopreservation of Mammalian Culture Cells: Preparation and Recovery of Samples Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4350

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

Protocol provides methods for cryofreezing and subsequent thawing of mammalian cells. Pre-confluent cells are trypsinized, pelleted, resuspended in freezing medium, and gradually frozen. When needed, frozen cells are thawed quickly under running tap water and transferred to growth medium. - [Read Cryopreservation of Mammalian Culture Cells: Preparation and Recovery of Samples Protocol]

Culture and Characterization of Human Embryonic Stem Cells. PDF - http://www.liebertonline.com/doi/pdf/10.1089/scd.2004.13.325

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: ES Cell Growth Media and Requirements

Culture and Characterization of Human Embryonic Stem Cells. DRAPER et al., STEM CELLS AND DEVELOPMENT 13:325Ã336 (2004) - [Read Culture and Characterization of Human Embryonic Stem Cells. PDF]

Culture and Expansion of OT-1 TCR Transgenic T cells Protocol - http://www.natureprotocols.com/2007/01/29/culture_and_expansion_of_ot1_t.php

Category: Immunology: T Cell Protocols

Protocol for culture and expansion of OT-1 TCR transgenic T cells. - [Read Culture and Expansion of OT-1 TCR Transgenic T cells Protocol]

Culture of BEND Cells (Bovine Endometrial Cells) Protocol - http://www.animal.ufl.edu/hansen/protocols/bend.htm

Category: Cell Culture Protocols

Protocol for culture of BEND cells (Bovine Endometrial Cells). Includes: Culture medium, Thawing Cells, Trypsinazation/ Splitting and freezing. - [Read Culture of BEND Cells (Bovine Endometrial Cells) Protocol]

Culture of Human Embryonic Stem Cells (hESC) - http://stemcells.nih.gov/research/NIHresearch/scunit/culture.asp

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture

Culture of Human Embryonic Stem Cells (hESC). one simple protocol outlined. NIH Stem Cell Unit - [Read Culture of Human Embryonic Stem Cells (hESC)]

Articles (1-10 of 24)

Lambda Phage Protocol Large DNA Preparation

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.

Genomic DNA Labeling of Microarrays Protocol

DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the labeling of genomic DNA for use as a probe for hybridization to the cDNA spotted on the array.

Drosophila Cell Transfection Protocol

A simple Drosophila tissue culture cell transfection method.

Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.

Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

This Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA protocol describes the production of probes labeled with the fluorescent dyes, Cy3 and Cy5, following the synthesis of cDNA from human mRNA and the hybridization of the probes to DNA microarrays.

Acid Guanidinium Thiocyanate RNA Isolation Phenol Chloroform Extraction

A single step RNA isolation protocol using Phenol Chloroform Extraction and Acid Guanidinium Thiocyanate. This RNA isolation method uses the fact that guanidinium thiocyanate can simultaneously lyse the cells and inactive cellular RNAses during the initial RNA isolation step allow a single step in the method.

Microinjection Pipettes Protocol

Microinjection Pipettes Preparation Protocol.

Acid Washing Microinjection Pipettes Protocol

Acid Washing of Glass microinjection pipettes protocol.

Paraffin Embedding Protocol

Paraffin Embedding Protocol for molecular profiling. This Paraffin Embedding Protocol describes the processing of the tissues into sections following ethanol fixation. Molecular profiling (MP) is a technique that is used to visualize the global patterns of RNA expression or protein expression in various cell types and disease processes.

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