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Cell Cycle Protocol using Flow Cytometry - http://www.people.cornell.edu/pages/jls269/protocols/cellcycleprotocols.html

Category: Cell Biology and Cell Culture: Cell Cycle Protocols

Cell Cycle Protocol using Flow Cytometry. Cell cycle Analysis by Flow Cytometry. DNA content Analysis on the FACSAria. Analyzing Cell cycle FCM data. Biomedical Sciences Flow Cytometry Core Laboratory. Dr.Smith Cornell. - [Read Cell Cycle Protocol using Flow Cytometry]

Cell Synchronization Using Centrifugal Elutriation Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4490

Category: Cell Biology and Cell Culture: Cell Cycle Protocols

protocol describes a method for the synchronization of cell populations using centrifugal elutriation. The method relies on the fact that cell size increases linearly as cells proceed through the cell cycle. Cells of similar size (and cell cycle phase) are eluted stepwise from the cell chamber, with the smallest size (those in early G1) being eluted first. Using this procedure, it is possible to obtain relatively pure populations of cells at various points in G1, S, and G2/M. - [Read Cell Synchronization Using Centrifugal Elutriation Protocol]

Cell Synchronization Using Centrifugal Elutriation Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4490

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Cycle Analysis Protocols

This protocol describes a method for the synchronization of cell populations using centrifugal elutriation. The method relies on the fact that cell size increases linearly as cells proceed through the cell cycle. Cells of similar size (and cell cycle phase) are eluted stepwise from the cell chamber, with the smallest size (those in early G1) being eluted first. Using this procedure, it is possible to obtain relatively pure populations of cells at various points in G1, S, and G2/M. - [Read Cell Synchronization Using Centrifugal Elutriation Protocol]

Chambers for Examination of Live Cells under Mechanical Stress Protocol - http://www.cshprotocols.org/cgi/content/extract/2007/3/pdb.ip32

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]

Characterization of Protein Glycosylation Using Chip-Based Nanoelectrospray with Precursor Ion Scann - http://jbt.abrf.org/cgi/content/full/16/3/209

Category: Molecular Biology Protocols: Carbohydrate Protocols

Characterization of Protein Glycosylation Using Chip-Based Nanoelectrospray with Precursor Ion Scanning Quadrupole Linear Ion Trap Mass Spectrometry. Sheng Zhang1 and Brian L. Williamson. Journal of Biomolecular Techniques, 16:209-219 2005. - [Read Characterization of Protein Glycosylation Using Chip-Based Nanoelectrospray with Precursor Ion Scann]

Chemical Sequencing Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3804

Category: DNA Protocols: DNA Sequencing Protocols

Protocol for chemical sequencing. - [Read Chemical Sequencing Protocol]

Chlorophyll Content Protocol - http://homepages.gac.edu/~cellab/chpts/chpt8/ex8-2.html

Category: Plant Biology Protocols: Photosynthesis and Respiration Protocols

Protocol describing how to calculate chlorophyll content. - [Read Chlorophyll Content 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 in Yeast Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4177

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Chromatin immunoprecipitation is a method for determining whether a particular protein binds to a particular DNA sequence in vivo. - [Read Chromatin Immunoprecipitation in Yeast Protocol]

Cloning into Bacteriophage M13 Vectors Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4018

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol describes three standard methods to construct bacteriophage M13 recombinants: (1) ligating insert DNA to a linearized vector, prepared by cleavage of M13 RF with a single restriction enzyme; (2) using alkaline phosphatase to suppress self-ligation of the linearized vector, and (3) using M13 RF cleaved with two restriction enzymes for directional cloning. - [Read Cloning into Bacteriophage M13 Vectors Protocol]

Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3835

Category: PCR Protocols: PCR Cloning Protocols

Pairs of oligonucleotide primers used in PCR are often designed with restriction sites in their 5' regions. In many cases, the sites are different in the two primers. In this case, amplification generates a target fragment whose termini now carry new restriction sites that can be used for directional cloning into plasmid vectors. The purified fragment and the vector are digested with the appropriate restriction enzymes, ligated together, and transformed into E. coli. - [Read Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA Protocol]

Cloning PCR Products into T Vectors Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3834

Category: PCR Protocols: PCR Cloning Protocols

This method of direct cloning takes advantage of the unpaired adenosyl residue added to the 3' terminus of amplified DNAs by Taq and other thermostable polymerases. - [Read Cloning PCR Products into T Vectors 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 Two-Cell- to Compacted Morula-Stage Embryos Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4359

Category: Stem Cell Protocols: Stem Cell Injection Protocols

This protocol describes a method for collecting two-cell- to compacted morula-stage embryos. Such embryos can then be used for microinjection. - [Read Collecting Two-Cell- to Compacted Morula-Stage Embryos 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 Early Mouse Embryos for RNAi Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4510

Category: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes a method to collect early embryos from 6-week-old mice. Subsequently, the isolated embryos can be injected with double-stranded RNA to induce knockdown of a gene of interest. - [Read Collection of Early Mouse Embryos for RNAi Protocol]

Collection of Mouse Oocytes for RNAi Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4509

Category: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes a method to collect oocytes from 6-week-old mice. Subsequently, the isolated oocytes can be injected with double-stranded RNA to induce knockdown of a gene of interest. - [Read Collection of Mouse Oocytes for RNAi Protocol]

Colony PCR Protocol II - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4141

Category: PCR Protocols: Colony PCR

Protocol describes the use of PCR to screen for bacteria that carry recombinant plasmids. The PCR can be carried out using the same primers as for amplification of the cloned insert. To determine the orientation of the insert, a third, insert-specific primer that is asymmetrically distanced from the clonal insertion site can be used. - [Read Colony PCR Protocol II]

Competitive RT-PCR: Estimation of Copy Number Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4111

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

In this protocol, sample and competitor RNAs are reverse transcribed (separately) in a pilot experiment. A constant amount of sample RT product is then combined with a 2-logserial dilution of competitor RT product for PCR. Procedure provides an approximate copy number for the sample, which is then fine-tuned by repeating the experiment with a series of twofold dilutions of competitor. The experiment includes controls for sample-to-sample variations in RT efficiency. - [Read Competitive RT-PCR: Estimation of Copy Number Protocol]

Competitive RT-PCR: Preparation of Competitor RNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4110

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

The first step in competitive RT-PCR is the synthesis and purification of the synthetic competitor. This is an RNA molecule designed to be reverse-transcribed and PCR-amplified with the same efficiency as the endogenous transcript of interest. Once the competitor molecule has been prepared, as described in this protocol, competitive PCR can be carried out. - [Read Competitive RT-PCR: Preparation of Competitor RNA Protocol]

Concentrating and Desalting Nucleic Acids with Microconcentrators Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4457

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]

Configuration Column Construction Column Packing for Capillary Liquid Chromatography - http://cshprotocols.cshlp.org/cgi/content/abstract/2007/3/pdb.prot4578

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol for configuration, column construction, and column packing for a capillary liquid chromatography system. Protocol describes a procedure for adapting conventional HPLC systems to provide accurate low-flow rates (0.4-4 µl/min) and gradients required to operate slurry-packed capillary columns. A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to several commercial UV detectors. - [Read Configuration Column Construction Column Packing for Capillary Liquid Chromatography]

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]

Construction of cDNA Libraries Stage 5: Fractionation of cDNA by Gel Filtration through Sepharose - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3730

Category: DNA Protocols: cDNA Library Protocols

Protocol for construction of cDNA library, stage 5: Fractionation of cDNA by Gel Filtration through Sepharose CL-4B. - [Read Construction of cDNA Libraries Stage 5: Fractionation of cDNA by Gel Filtration through Sepharose]

Construction of cDNA Libraries in Eukaryotic Expression Vectors for Construction and Screening - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4068

Category: DNA Protocols: Genomic DNA Library Protocols

This stage summarizes how to construct a cDNA library in a mammalian vector, using commercial kits. These kits are available from several manufacturers and generally include all the required reagents. - [Read Construction of cDNA Libraries in Eukaryotic Expression Vectors for Construction and Screening]

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In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.

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