content Protocols

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]

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]

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]

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]

Category: Protein Protocols: Protein Extraction From Tissue

Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections. Ikedaa et al, 1998 JHC. - [Read Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tis]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

Protocol is the first in a set of three describing fluorescent mRNA differential display (FDD or FDDRT-PCR).  The method begins with the harvesting of total RNA from blood samples. - [Read Extraction and Purification of RNA from Blood Samples for Fluorescent mRNA Differential Display]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

Protocol is the first in a set of three describing fluorescent mRNA differential display (FDD or FDDRT-PCR).  The method begins with the harvesting of total RNA from tissue samples. - [Read Extraction and Purification of RNA from Tissue Samples for Fluorescent mRNA Differential Display]

Category: PCR Protocols

Protocol is the first in a set of three describing fluorescent mRNA differential display (FDD or FDDRT-PCR).  The method begins with the harvesting of total RNA from the tissue-cultured cells of interest.  For other starting materials, such as blood samples, please see Extraction and Purification of RNA from Blood Samples for Fluorescent mRNA Differential Display. - [Read Extraction and Purification of RNA from Tissue-Cultured Cells for Fluorescent mRNA Differential]

Category: Bacterial Cell Culture Protocols

The growth conditions of microbial cell cultures and the time of sample collection should be optimized and standardized when growing cells for protein extraction. Because cells may excrete proteases and other extracellular enzymes, and compounds in the medium may interfere with extraction, wash cultures with an isotonic buffer, such as PBS or sucrose before solubilization. - [Read Extraction and Solubilization of Total Protein from Microorganisms Protocol]

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

Protocol on the extraction and solubilization of total protein from plant seeds. - [Read Extraction and Solubilization of Total Protein from Plant Seeds Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Formamide can be used instead of proteinase K to dissociate bacteriophage {lambda} coat proteins from the viral {lambda}. The procedure is rapid and works best with large-scale preparations of bacteriophage {lambda}. - [Read Extraction of Bacteriophage lambda DNA from Large-scale Cultures Using Formamide Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

DNA is best isolated from bacteriophage lambda particles by digesting the viral coat proteins with a powerful protease such as proteinase K, followed by extraction with phenol:chloroform. This procedure is easily adapted for use with smaller-scale preparations of bacteriophage lambda. - [Read Extraction of Bacteriophage lambda DNA from Large-scale Cultures Using Proteinase K and SDS Protocol]

Category: Plant Biology Protocols: Arabidopsis Protein Peptide Protocols

The simplest way to analyze proteins is in unfractionated extracts. However, it is often desirable to fractionate proteins, e.g, by size. This procedure extracts total protein from Arabidopsis samples. Typical yields are ~2-3 mg/ml (using rosette leaves) or 6-8 mg/ml (using young seedlings). - [Read Extraction of Total Protein from Arabidopsis Protocol]

Category: PCR Protocols

Protocol for FDD PCR, identification and analysis of differentially expressed cDNAs. - [Read FDD PCR, Identification and Analysis of Differentially Expressed cDNAs Protocol]

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol provides methods for fixation of mouse embryos and tissues with paraformaldehyde, Bouin’s fixative, or methanol/DMSO solution. - [Read Fixation of Mouse Embryos and Tissues Protocol]

Category: Histology Protocols: Fixation of Cells Tissues Protocol

Analysis of Cell Cycle Based on DNA Content. FIXATION PROTOCOL FOR FLOW CYTOMETRIC ... Liberate cells from tissue culture flask by trypsin digestion and ... FIXATION PROTOCOL FOR FLOW CYTOMETRIC ANALYSIS OF DNA BY PI STAINING. Auburn University, College of Veterinary Medicine. You. - [Read FIXATION PROTOCOL FOR FLOW CYTOMETRIC ANALYSIS OF DNA BY PI STAINING]

Category: Cell Culture Protocols: Cell Fixing Protocols

Organic solvents such as alcohols and acetone remove lipids and dehydrate cells, precipitating the proteins on the cellular architecture. Be aware that different antigens may be affected differently by the various solvents. If no previous data are available for your antigen, start with the 50/50 mixture. For tissue culture dishes, concentrations of acetone higher than 50% will destroy the integrity of the plastic. - [Read Fixing Attached Cells in Organic Solvents Protocol]

Category: Cell Culture Protocols: Cell Fixing Protocols

Treating cells with paraformaldehyde leads to the establishment of chemical cross-links between free amino groups. When the cross-links join different molecules, a latticework of interactions occurs that holds the overall architecture of the cell together. Commercial formaldehyde solutions are not recommended, because they lack the advantages of using a variable-length polymer, and the cells will simultaneously be fixed with the alcohol (usually methanol). - [Read Fixing Attached Cells in Paraformaldehyde Protocol]

Category: C. elegans Protocols: C. elegans Fixing Protocols

Bouin’s fixative is a particularly good choice for worms because it penetrates dense tissues well and is extremely good for fixing antigens. Like all strong fixatives, however, it is unsuitable for some antibody-antigen pairs. In such cases, the length of time in the Bouin’s fixative can be shortened, or paraformaldehyde fixation can be used instead. - [Read Fixing Caenorhabditis elegans in Bouin’s Fixative Protocol]

Category: C. elegans Protocols: C. elegans Fixing Protocols

Common method for fixing worms is to use paraformaldehyde. This method provides a gentler fixation than the Bouin’s method, but often requires the use of collagenase. This method is particularly good for examining adult worms. - [Read Fixing Caenorhabditis elegans in Paraformaldehyde Protocol]

Category: Cell Culture Protocols: Cell Fixing Protocols

Treating cells with paraformaldehyde leads to the establishment of chemical cross-links between free amino groups. When the cross-links join different molecules, a latticework of interactions occurs that holds the overall architecture of the cell together. - [Read Fixing Suspension Cells with Paraformaldehyde Protocol]

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)]

Category: Microscopy Protocols: Fluorescence Microscopy Protocols

Most biological specimens are relatively transparent, so details of internal and intracellular morphology are difficult to image in untreated living specimens using simple bright-field techniques. Fluorescence microscopy offers greater advantages and possibilities for increasing contrast and determining the specific localization of molecules in cells. Article outlines the three methods most commonly used to introduce an appropriate label into Drosophila tissue without perturbing the process. - [Read Fluorescent Reagents for Live Cell Imaging and Their Introduction into Cells]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Immunofluorescence Staining Protocols

Protocol provides a method for analysis of cell surface antigens using direct immunofluorescence, i.e., when the antibody being used is directly labeled to a fluorescent dye. After staining, the cells can be analyzed immediately by flow cytometry or fixed and stored for later analysis. - [Read Fluorescent Staining of Cell Surface Antigens on Viable Cells Using Direct Immunofluorescence]

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]