conditions Protocols

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

Protocol is used to establish conditions for restriction enzyme digestion of eukaryotic genomic DNA that will generate fragments of a size appropriate for construction of genomic libraries.  To construct a genomic library, the average length of the starting genomic DNA should be at least eight times the capacity of the vector. - [Read Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Pilot Reactions Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

The results of the pilot experiment (Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Pilot Reactions) are used to establish conditions for partial digestion of eukaryotic DNA in the large-scale reactions described here. - [Read Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Preparative Reactions Protocol]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: PCR Optimization Protocols

A Great PCR and Multiplex Guide. Topics for PCR optimization. Primer conditions, PCR temperatures, etc. Octavian Henegariu. - [Read PCR and Multiplex Guide]

Category: PCR Protocols: PCR Optimization

A Great PCR and Multiplex Guide. Topics for PCR optimization. Primer conditions, PCR temperatures, etc. Octavian Henegariu. - [Read PCR and Multiplex Guide]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Peroxisome Isolation Protocols

To isolate peroxisomes from Saccharomyces cerevisiae of a quality sufficient for in vitro import studies, we optimized the conditions for cell growth and for cell fractionation. Stability of the isolated peroxisomes was monitored by catalase latency and sedimentability of marker enzymes. - [Read Peroxisomes in Saccharomyces cerevisiae]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: PCR Optimization Protocols

Optimizing PCR Reaction Conditions. Optimizing PCR Tips and Guide. University of Alaska Museum - [Read Polymerase Chain Reaction (PCR) Optimization]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol describes a general approach for the direct attachment of a triazine dye onto a carbohydrate support.  The direct coupling method is achieved under alkaline conditions (pH 10-11) with nucleophilic displacement of the dye’s chlorine atom by the polymer hydroxyls. - [Read Preparation of Affinity-Ligand Resins by Immobilization of Dyes on Polyhydroxyl Matrices Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

This rapid method is used to screen bacteriophage {lambda}gt11 clones for the production of immunodetectable fusion proteins. After optimizng the conditions of infection and induction, the method can be used to produce preparative amounts of a fusion protein. - [Read Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage {lambda}: Lytic Infection]

Category: Immunology: T Cell Protocols

Protocols describe the conditions required to induce proliferation are described. Also describe the assay in which CD4·CD25·T cells are co-cultured with conventional T cells in order to assess their suppressive function. Will describe the culture conditions for the activation and expansion of CD4·CD25· cells. - [Read Proliferative Assays for T Cell Function Protocols]

Category: Plant Biology Protocols: Arabidopsis Protein Peptide Protocols

Coimmunoprecipitation is one of the most useful techniques for revealing protein-protein interactions. Good negative controls to verify the specificity of the coimmunoprecipitation procedure are (1) performing the same immunoprecipitation experiment using beads coupled to the preimmune serum and (2) probing the Western blot with antibodies against protein known not to interact with the coimmunoprecipitated proteins under physiological conditions. - [Read Protein Coimmunoprecipitation in Arabidopsis Protocol]

Category: Signalling Signal Transduction Protocols

Protocol for immunopurification of tyrosine phosphorylated proteins. Protocol includes: Cell Lysis (Non-Denaturing and Denaturing Conditions) and Purification. - [Read Protocol for Immunopurification of Tyrosine Phosphorylated Proteins]

Category: Cell Biology and Cell Culture

This protocol focuses on the interactions between L-selectin expressed on neutrophils and PNAd coated onto the plastic surface. The main purpose of the flow chamber assay is to visualize and measure interactions between flowing cells expressing a given adhesion molecule on their surface, and their receptor, either directly coated on the flow chamber lower wall or expressed on a cell monolayer. - [Read Protocol for L-selectin-PNAd Interactions under Flow Conditions.]

Category: Cell Biology and Cell Culture

This protocol introduces the use of a liquid-filled wash chamber that separates unbound cells by gravity thereby eliminating uncontrolled shear forces and passage of adherent cells through a liquid/air interface. The cells are loaded with a fluorescent dye (6-carboxyfluorescein diacetate) for detection although other methods such as radioactive labels malabels may be used. This protocol is also useful for assaying molecules that promote or inhibit cell adhesion. - [Read Protocol for Measurement of Cell Adhesion Under Static Conditions]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: Standard PCR Protocol

Avoiding Contamination, Preparation of Reaction Mixture, and Cycling Conditions of a PCR Protocol - Fermentas - [Read Protocol for PCR with Taq DNA Polymerase - Fermentas]

Category: PCR Protocols: Standard PCR Protocol

Avoiding Contamination, Preparation of Reaction Mixture, and Cycling Conditions of a PCR Protocol - Fermentas - [Read Protocol for PCR with Taq DNA Polymerase - Fermentas]

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

Includes protocols: Mouse Embryonic Fibroblasts (MEF) Preparation; Harvesting MEFs; Cryopreservation of MEFs; Thawing and maintaining MEFs; Irradiating & Plating MEFs; Culture of Human ES cells with Matrigel® and Conditioned Medium; Preparation of Conditioned Medium (CM); Preparation of Matrigel® -coated plates; Passage of human ES cells on Matrigel®; Daily maintenance of feeder-free culture; Freezing Human ES Cells; Thawing Human ES cells; Formation of Embryoid Bodies; - [Read Protocols for the Maintenance of Human Embryonic Stem Cells in Feeder Free Conditions]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Immobilized metal-ion affinity chromatography (IMAC) is suitable for the purification of proteins under denaturing conditions.  Either guanidine-HCl or urea can be used, although guanidine-HCl is a stronger denaturant than urea.  Proteins that have been adsorbed to the column in the presence of guanidine-binding buffer may be washed with urea-binding buffer and eluted with urea elution buffer. - [Read Purification of Histidine-Tagged Proteins under Denaturing Conditions Using IMAC Protocol]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Histidine-tagged proteins can be purified on prepacked 1-ml immobilized metal-ion affinity chromatography (IMAC) columns without optimization of the separation conditions.  The method allows fast capture of the target protein, although with a lower purity than can be obtained under optimized conditions. - [Read Purification of Histidine-Tagged Proteins Using IMAC Without Parameter Optimization Protocol]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Mitochondria Isolation Protocols

A procedure developed to purify simultaneously peroxisomes and mitochondria from spinach (Spirnacia olercea L.) leaf under isoosmotic and low viscosity conditions. This method involves differential centrifugation and density gradient centrifugation on four layers of Percoll. - [Read Purification of Peroxisomes and Mitochondria from Spinach Leaf by Percoll Gradient Centrifugation]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Peroxisome Isolation Protocols

A procedure developed to purify simultaneously peroxisomes and mitochondria from spinach (Spirnacia olercea L.) leaf under isoosmotic and low viscosity conditions. This method involves differential centrifugation and density gradient centrifugation on four layers of Percoll. - [Read Purification of Peroxisomes and Mitochondria from Spinach Leaf by Percoll Gradient Centrifugation]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol describes a general method for the purification of protein using dye-ligand affinity chromatography.  The effectiveness of this protocol is dependent upon the reader having previously determined the optimal dye and buffer conditions to use for binding and eluting the protein(s) of interest. - [Read Purification of Protein Using Dye-Ligand Affinity Chromatography Protocol]

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

Using excitation at 365 nm and measuring emission at 455 nm, the amount of 4-MU produced can be quantified. Under these conditions, background fluorescence from the substrate is negligible, especially if the appropriate filter is selected. - [Read Quantitative GUS Activity Assay of Plant Extracts]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Plaques formed by M13 bacteriophages or bacterial colonies transformed by plasmids carrying specific mutations can be detected by hybridization, using a radiolabeled oligonucleotide that forms a perfect duplex with the mutant sequence. Hybridization is carried out under conditions of low stringency that allow the radiolabeled oligonucleotide to anneal to both mutant and wild-type DNAs. - [Read Screening Recombinant Clones for Site-directed Mutagenesis by Hybridization to Radiolabeled Oligos]

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

Protocol for single tube confirmation yeast PCR. Includes: Clonal purification; Zymolyase treatment; PCR reactions; PCR conditions; Agarose gel electrophoresis. - [Read Single Tube Confirmation Yeast PCR Protocol]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol describes how to perform a test separation using a set of protein standards that have been designed to evaluate column performance. - [Read Standard Chromatographic Conditions for RP-HPLC of Proteins Protocol]