presence Protocols

Category: PCR Protocols: PCR Optimization

Procedure details the establishment of an amplification procedure for GC-rich sequences.  The DNA fragments of interest are amplified in the presence of either 5% DMSO, 1 M betaine, 2 M betaine, 1 M betaine, and 5% DMSO; 2 M betaine and 5% DMSO; 0.4 M tetramethylene sulfone; or without any of the enhancers. - [Read PCR Amplification of Highly GC-Rich Regions Protocol]

Category: Bacteria Genetics Protocols

Protocol describes a method to determine the presence of plasmid DNA in an Agrobacterium culture. Compared to selection of transformed Agrobacterium, which can be ambiguous and normally takes several days for resistant colonies to appear, the approach described here is both rapid and accurate. - [Read PCR Analysis of Agrobacterium Protocol]

Category: Signalling Signal Transduction Protocols

Protocol describes a competitive ligand binding assay for cortical neurotrophin receptors. Following binding in the presence of competitor, the bound radiolabeled ligand is cross-linked to the receptor. The cells are lysed and the ligand-receptor complexes are immunoprecipitated using a pan-trk (tyrosine kinase receptor) antibody. Protocol includes:Preparation of Cortical Tissue for Competitive Crosslinking, Competitive Binding, Crosslinking Ligand to Receptor, Lysis and Immunoprecipitation etc - [Read Protocol for Competitive Ligand Binding to Cortical Receptor using Crosslinking]

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: PCR Protocols: Quantitative PCR Protocols

Protocol that uses the polymerase chain reaction (PCR) to quantitate the numbers of a particular DNA sequence, from 1 to 20,000 molecules per sample.  In addition, it helps assess the presence of contaminating sequences, which can seriously affect the outcome of the procedure. - [Read Quantitation of Rare DNAs by PCR Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

In this test rabbit articular chondrocytes are cultured in the presence of test compound, the toxicity of which is then determined by its effect on the production of proteoglycan by the cells, as detected by the dye Alcian Blue. - [Read Rabbit Articular Chondrocyte Functional Toxicity Test]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

Protocol for radiolabeling of DNA by extension of random oligonucleotides in the presence of melted agarose. - [Read Radiolabeling of DNA by Extension of Random Oligonucleotides in the Presence of Melted Agarose]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this procedure, synthesis of cDNA is carried out in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP.  After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of E. coli DNA polymerase. - [Read Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

In the first part of this protocol, the linear range of amplification is determined by carrying out 10 identical PCRs in the presence of [{alpha}-32P]dCTP and stopping one reaction after every two cycles.  Amplification products are quantified on a denaturing polyacrylamide gel and the results plotted on a graph (counts per minute vs. cycle number).  Total RNA is used as an internal control. - [Read Relative RT-PCR: Determining the Linear Range of Amplification and Optimizing the Primers:Competimer]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for restriction endonuclease digestion of DNA in agarose plugs. Genomic DNA isolated from mammalian, yeast, or bacterial cells can be digested with restriction endonucleases by incubating agarose plugs containing the DNA in the presence of the desired enzyme.  After digestion, the DNA can be fractionated by pulsed-field gel electrophoresis and either isolated from the gel or analyzed by Southern Hybridization. - [Read Restriction Endonuclease Digestion of DNA in Agarose Plugs Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

A powerful way to identify a mutation in the gene of interest and to test mutant plants for phenotypes that are predicted to result from loss of function of that gene is by PCR screening. Pools of insertion lines are screened using one primer corresponding to the gene of interest and one primer corresponding to the end of the insertion element. The synthesis of a product indicates the presence of an insertion in the gene of interest. - [Read Screening DNA Pools for T-DNA Insertions in Arabidopsis Genes Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

In an attempt to accurately measure DNA content with simultaneous preservation of cell surface markers, we have utilized gentle ethanol treatment techniques, which permeablize cells with minimal loss of surface antigen expression and antibody-antigen association. For some cell types, the presence of apoptotic cells based on reduced DNA content can also be detected. One such technique employs the addition of ethanol to cells previously resuspended in high concentrations of fetal bovine serum... - [Read Simultaneous Analysis of DNA Content and Surface Immunophenotype Protocol]

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

Rabbit-derived corneal cells are cultured in the presence of test compounds, the toxicity of which are determined by their effect upon cell viability.  A decrease in cell number, as measured by uptake of the dye Neutral Red, serves as an indicator of potential cytotoxicity.  This test has been proposed as a potential replacement alternative for the Draize Eye Irritation test. - [Read SIRC Cytotoxcitiy Test]

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

Demonstrates the use of the FACSort for sorting based on DNA ploidy or the presence of cytokeratin, and the suitability of the sorted cells for morphologic analysis by microscopic examination. - [Read Sorting Aberrant Cells Using Anti-Cytokeratin or DNA Content for Study of Cell Morphology]

Category: Immunology: Mononuclear Cell Isolation Protocols: T-Cell Isolation Protocols

Protocol describes removal of T cell subsets by cytotoxic elimination using CD-specific antibodies. Describes the complete removal of T cells from lymphocyte preparations based on the presence of the glycoprotein Thy-1 on the cell surface of T lymphocytes. Cytotoxic elimination is employed; however, Thy-1-specific antibodies are used rather than MHC class II-specific antibodies so that T cells are eliminated rather than B cells and accessory cells. - [Read T Cell Depletion by Cytotoxic Elimination Protocol]

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

This method for yeast colony PCR is used to test for the presence of target sequences in cells. - [Read Yeast Colony PCR]

Category: PCR Protocols: Colony PCR

Protocol for yeast colony PCR. This method for yeast colony PCR is used to test for the presence of target sequences in cells. - [Read Yeast Colony PCR Protocol]