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Disaggregating Cleavage-Stage Embryos and the Inner Cell Mass of Blastocysts into Individual Cells - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4427

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol describes a method for disaggregating embryos and the ICM of blastocysts into individual cells. - [Read Disaggregating Cleavage-Stage Embryos and the Inner Cell Mass of Blastocysts into Individual Cells]

Dissection of Spinal Cords for Analysis of Axonal Pathfinding in dsRNA Treated Avian Embryos - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4505

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes how to dissect spinal cords of dsRNA-treated avian embryos for analysis of commissural axon guidance. After fixation of the spinal cord with paraformaldehyde, commissural axons are stained with Fast-DiI. - [Read Dissection of Spinal Cords for Analysis of Axonal Pathfinding in dsRNA Treated Avian Embryos]

Dissection of Spinal Cords for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4505

Category: Avian Bird Protocols

DNA Electroelution - http://rothlab.ucdavis.edu/protocols/dna-electroelution.html

Category: DNA Protocols: DNA Extraction Protocols: DNA Extraction Polyacrylamide Gels

DNA Electroelution. This protocol describes the purification of DNA by trapping in a high-salt cushion in a "UEA AnalyticalElectroeluter" (IBI). This machine is no longer manufactured, to our knowledge. However, a smiliar device can be easily made from Plexiglas according to the following diagram, taken from Cornel Mulhardt, Molecular Biology and Genomics (2007) Academic Press, p.52: Schimenti Lab - [Read DNA Electroelution]

DNA Extraction from Agarose Gels Protocol - http://www.methodbook.net/dna/gelextrc.html

Category: DNA Protocols: DNA Extraction Protocols: Agarose Gel DNA Extraction Protocols

DNA Extraction from Agarose Gels Protocol. The page includes cutting out the DNA band from the gel, and describes three methods including 1) Spin-columns (Nucleic acid purification columns), 2) using Dialysis tubing (semi-permeable membrane, Visking tubing), and the 3) Paper strip method.Matt Lewis, Department of Pathology, University of Liverpool. - [Read DNA Extraction from Agarose Gels Protocol]

DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_124-126.pdf

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol describes here a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. Includes: In situ hybridization with DIG-labeled probes; Detection of DIG-labeled probes; Fluorescence microscopy. - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System]

DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_124-126.pdf

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol describes a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. This procedure can be used to detect single copy sequences as small as 1 kb on human metaphase chromosomes. - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol]

Dye Terminator Cleanup for Automated DNA Sequencing Reactions Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4101

Category: DNA Protocols: DNA Sequencing Protocols

Protocol describes a simple procedure for purifying sequencing reactions using the MagneSil particles from Promega. - [Read Dye Terminator Cleanup for Automated DNA Sequencing Reactions Protocol]

Electroporating DNA into Embryonic Stem (ES) Cells and Selection Methods Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4409

Category: Mouse Protocols: Mouse Genetics Protocols

Protocol describes a method for electroporating DNA into ES cells, as well as selection methods. Pilot studies should be performed to optimize the conditions for each DNA construct. The selection method described here is one of the most complex. It involves targeting constructs in which the bacterial neomycin-resistance gene disrupts the coding sequence of the mouse gene. - [Read Electroporating DNA into Embryonic Stem (ES) Cells and Selection Methods Protocol]

ELISA Method Measure Inhibition COX Enzymes - http://www.natureprotocols.com/2006/11/22/an_elisa_method_to_measure_inh.php

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]

ELISA Method to Measure Inhibition of the COX Enzymes Protocol - http://www.natureprotocols.com/2006/11/22/an_elisa_method_to_measure_inh.php

Category: ELISA Protocols

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This 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. - [Read ELISA Method to Measure Inhibition of the COX Enzymes Protocol]

Enrichment Of Monocytes For Morphological Study Protocol - http://www.bdbiosciences.com/pdfs/whitePapers/23-2785-01.pdf

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Sorting Protocols

Protocol describes how monocytes can be sorted from a lysed whole blood sample, and that cytospin preparations can be made for examination by light microscopy. - [Read Enrichment Of Monocytes For Morphological Study Protocol]

Epstein-Barr Virus Transformation of Lymphoblasts Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4481

Category: Immunology

Protocol for Epstein-Barr Virus transformation of lymphoblasts. Protocol describes a method for the transformation of lymphoblasts by Epstein-Barr Virus (EBV). Cells may be isolated from whole blood or taken from cryopreserved, non-immortalized stocks. - [Read Epstein-Barr Virus Transformation of Lymphoblasts Protocol]

Estimation of Cell Number by Hemocytometry Counting Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4454

Category: Cell Culture Protocols

Protocol describes a method for estimation of mammalian cell number in a defined volume of medium using a hemocytometer. Automated methods using cell-counting devices such as those produced by Coulter are desirable when large numbers of individual samples are to be counted. - [Read Estimation of Cell Number by Hemocytometry Counting Protocol]

Ethyl Methane Sulfonate (EMS) Mutagenesis Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4180

Category: Yeast Protocols: Yeast Genetics Protocols

Protocol describes mutagenesis of yeast with ethyl methane sulfonate (EMS). It causes approximately 40-70% cell death in most haploid laboratory strains, a level of cell killing that is commonly used in mutant hunts with haploid strains. - [Read Ethyl Methane Sulfonate (EMS) Mutagenesis Protocol]

Exon Trapping and Amplification for Construction of the Library Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4075

Category: DNA Protocols: Genomic DNA Library Protocols

Protocol describes how to construct and amplify a genomic DNA library in a plasmid vector (pSPL3) equipped to express cloned exons in transfected mammalian cells. - [Read Exon Trapping and Amplification for Construction of the Library Protocol]

Expression of Cloned Genes in E. coli Using IPTG-inducible Promoters Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4085

Category: E Coli Protocols: E coli Protein Protocols

Protocol for the expression of cloned genes in E. coli using IPTG-inducible promoters. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying IPTG-inducible promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using IPTG-inducible Promoters Protocol]

Expression of Cloned Genes in E. coli Using the Bacteriophage lambda pL Promoter Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4086

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage {lambda} pL promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage lambda pL Promoter Protocol]

Expression of Cloned Genes in E. coli Using the Bacteriophage lambda pL Promoter Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4086

Category: E Coli Protocols: E coli Protein Protocols

Protocol for expression of cloned genes in E. coli using the bacteriophage lambda pL promoter. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage lambda pL promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage lambda pL Promoter Protocol]

Expression of Cloned Genes in E. coli Using the Bacteriophage T7 Promoter Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3597

Category: E Coli Protocols: E coli Protein Protocols

Protocol for the expression of cloned genes in E. coli using the bacteriophage T7 promoter. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage T7 promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage T7 Promoter Protocol]

Extraction of Calf Brain Lipids Protocol - http://www.bio.com/protocolstools/protocol.jhtml?id=p1281

Category: Lipid Protocols

Protocol for the extraction of calf brain lipids. Protocol describes a rapid method to isolate lipids from bovine brain tissue using an organic solvent mixture of Chloroform and Methanol. - [Read Extraction of Calf Brain Lipids Protocol]

Fabrication Protocol for DNA Microarrays - http://research.nhgri.nih.gov/microarray/fabrication.shtml

Category: DNA Microarray Protocols

This protocol describes the steps required to produce a cDNA microarray. Gene-specific DNA is produced by PCR amplification of purified template plasmid DNAs from cloned ESTs. The PCR product is purified by ethanol precipitation, thoroughly resuspended in - [Read Fabrication Protocol for DNA Microarrays]

Flow Cytometric Analysis of Mitochondrial Transmembrane Potential ({Delta}{Psi}m) - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4465

Category: Cell Organelle Protocols: Mitochondria Protocols

Protocol describes a method for evaluation of mitochondrial function using the fluorochrome CMXRos. CMXRos is sequestered by actively respiring mitochondria, but washed out when the mitochondrial membrane potential is lost. This analysis can be combined with the TUNEL technique or immunocytochemistry. - [Read Flow Cytometric Analysis of Mitochondrial Transmembrane Potential ({Delta}{Psi}m)]

Fluorometric Quantitation of DNA Using Hoechst 33258 Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4458

Category: Nucleic Acid Detection Protocols

Protocol describes the quantitation of DNA using Hoechst 33258, a fluorescent dye that binds to double-stranded DNA. Fluorometry is simple and more sensitive than spectrophotometry, and allows the detection of nanogram quantities of DNA. The assay can only be used to measure the concentration of DNAs whose sizes exceed ~1 kb, as Hoechst 33258 binds poorly to smaller DNA fragments. - [Read Fluorometric Quantitation of DNA Using Hoechst 33258 Protocol]

Fractionation of Maize Embryo Proteins for 2-D Gel Electrophoresis Using Multicompartment Electrolyz - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4235

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

Protocol describes a method for performing isoelectric fractionation of a maize embryo sample using a multicompartment electrolyzer(MCE). This prefractionation of proteins having pIs within a certain pH interval is essential for allowing high loads of protein to be resolved on narrow and ultra-narrow immobilized pH gradients used in 2D electrophoresis. The isoelectric membranes in the MCE act like isoelectric traps capturing all the protein species having pIs encompassing the pI value of each... - [Read Fractionation of Maize Embryo Proteins for 2-D Gel Electrophoresis Using Multicompartment Electrolyz]

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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.

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.

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.

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.

Constructing Random-Peptide Libraries for Phage Display

The protocol describes the construction of a large (10^8 to 10^10) primary random peptide phage library in fUSE5 or f88-4 strains.

Histone H1 Kinase Activity Assay

Histone H1 Kinase Activity Assay Protocol. This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography.

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.

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