results Protocols

Category: Signalling Signal Transduction Protocols

Technique for Alkaline Phosphatase (APAAP). Techniques includes information on: Cytological Preparations, Fixation, Technique, Results, Substrate Solution and 0.005M Tris/HC1 Saline Buffer pH 7.6. - [Read Alkaline Phosphatase (APAAP) Technique]

Category: Signalling Signal Transduction Protocols

Protocol on an image-based assay of endothelial cell tube formation as a model of angiogenesis. Includes: Introduction, Methods, Results, Discussion and References. - [Read An Image-Based Assay of Endothelial Cell Tube Formation]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

This protocol describes an in vitro transcription assay that allows for a single round of transcription from in vitro assembled chromatin. Comparing the activity of a receptor or transcriptional coactivator in an assay that measures only a single round of transcription with the results from multiple rounds of transcription can help elucidate the mechanism of transcriptional activation by those factors. - [Read Assay:Single Round of In Vitro Transcription from Assembled Chromatin Templates Using a HeLa Cell Ex]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol for In situ hybridization to human metaphase chromosomes using DIG-, biotin-, or fluorochrome-labeled DNA probes and detection with fluorochrome conjugates. Includes: Pretreatment of metaphase spreads on slides; Denaturation and hybridization; Single color fluorescent detection with immunological amplification; Multicolor fluorescence in situ hybridization (Multicolor FISH); Results obtained with human metaphase chromosome spreads. - [Read In Situ Hybridization to Human Metaphase Chromosomes using DIG-, Biotin- or Fluorochrome-Labeled DNA]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

The results of cytotoxicity tests in primary cultures of rat hepatocytes and in MDBK and McCoy cells can be used to predict the in vivo 4-wk maximum tolerated dose in rats and dogs.  A correlation between in vitro cytotoxicity, as measured in this system, and LD50 values in rats and mice has also been established. - [Read In Vitro Prediction of the Maximum Tolerated Dose Protocol]

Category: Tetrahymena Protozoa Protocols

Mature Tetrahymena cells of opposite mating types are starved under appropriate salt conditions. The mating types are then combined to costimulate through cell-cell interaction. Loose pairs and then firm, irreversible pairs of cells of opposite mating types form. This method consistently results in a high percentage of pairing (usually greater than 80%) and good synchrony. - [Read Induction of Conjugation in Tetrahymena Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Procedure is used to prepare DNA simultaneously from many different types of samples or tissues.  Although the DNA is generally too small (approx.  80 kb) for efficient construction of genomic DNA libraries, it gives excellent results in Southern hybridizations and PCRs.  Cultured aneuploid mammalian cells (2 x 107, e.g., HeLa cells) yield 100 µg of DNA in a volume of 1 ml. - [Read Isolation of DNA from Mammalian Cells by Spooling Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: B Lymphocyte Isolation Protocols

A flow cytometry technique is presented, which results in the selection and isolation of two populations of cells from a complex mixture based on physical properties (e.g., size and internal granularity) and correlated expression of several surface molecules - [Read Isolation of Ly-1+/CD5+ B Cells by Cell Sorting Protocol]

Category: Animal Research Techniques

Minimization of Animal Numbers Used to Obtain Valid Results. - [Read John Hopkins Animals Research Use and Care]

Category: PCR Protocols: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]

Category: Bacterial Protocols

Ice tea has a complex composition, which leads to reduced filterability, and a decrease in sample throughput. Its composition can generate background or false positive signals. It is also well known that ice tea contains molecules that can inhibit the bioluminescence reaction, which can generate false negative results. The aim of this study was to develop a protocol that was able to neutralize these affects and enable faster detection of contamination. - [Read Microbial Detection in Ice Tea Using the Millipore Milliflex Rapid Microbiology Detection System]

Category: Cytogenetics Protocols: Comparative Genomic Hybridization CGH Protocols

Based on the original protocol describing CGH by Kallioniemi et al. - [Read Molecular Analysis and Results - DNA Comparative Genomic Hybridization (CGH)]

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: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

A. thaliana has a very small haploid genome and this makes obtaining DNA somewhat difficult. The most notable problem is that DNA is usually contaminated with polysaccharide which inhibit restriction enzymes as well as other DNA modifying enzymes. This problem is most easily solved by using young plants which have not accumulated as much polysaccharide as older plants. The best results are obtained with plants that are two to three weeks post germinated. - [Read Plant DNA Extraction Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Polyacrylamide Gels Protocols

Protocol for purification of synthetic oligonucleotides by polyacrylamide gel electrophoresis. As a rule of thumb, oligonucleotides >25 nucleotides should be purified by polyacrylamide gel electrophoresis, as should oligonucleotides of any length that yield anomalous results.  After electrophoresis, the oligonucleotide is eluted from the gel and concentrated by reversed-phase chromatography on Sep-Pak C18 columns. - [Read Purification of Synthetic Oligonucleotides by Polyacrylamide Gel Electrophoresis Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Protocol to be used as reference to help standardization of both the isolation procedure and the quality of the cultures allowing a better comparison of the results obtained from different laboratories. - [Read Rat Chromaffin Cells Primary Cultures: Standardization and Quality Assessment for Single-Cell Assay]

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: General Research Protocols and Methods: Sterile Technique

Sterile technique is one of the most important steps in insuring consistent results when employing recombinant DNA and protein expression techniques. Dr. Chazin Lab, Center for Structural Biology, Vanderbilt Univ. - [Read Sterile Technique for DNA and Protein Expression]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

This protocol fixes and prepares embryos for in situ hybridization to visualize transcript expression patterns. It is a modification of the method developed by Tautz and Pfeifle for whole-mount in situ analysis of embryos. Use of the standard hybridization protocol on RNAi-treated embryos results in high background staining, which makes visualization of transcript expression patterns practically impossible. The following modifications eliminate this problem and allow visualization of transcript. - [Read Transcript In Situ Hybridization of Whole-Mount Embryos for Phenotype Analysis of RNAi-Treated]

Category: Transfection Protocols

The following procedure is for simultaneous transfection and plating of RAW 264.7 cells. This protocol results in approximately 50% to 70% cell viability, and of those viable cells, 20% to 40% are transfected when using pEYFP-N1 from Clontech. Include: Procedure for Splitting Cells before Transfection; Procedure for Preparing Lipofectamine 2000 and DNA; Preparation of RAW 264.7 Cells for Transfection. - [Read Transfecting and Plating RAW 264.7 Cells with Lipofectamine 2000 Protocol]

Category: Real Time PCR

Validation of Microarray Results Using Quantitative Real-Time PCR (QRT PCR) with Omniscript RT. Dr. Craig Cadwell - [Read Validation of Microarray Results Using Quantitative Real-Time PCR (QRT PCR) with Omniscript RT]