Concentration of DNA by Ethanol Precipitation Protocol. Adapted fromBruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma. Usually 2.5 - 3 volumes of ethanol and/or acetate solution is added to the DNA in a microcentrifuge tube. This is then put into an ice-water bath for at least 10 minutes. The precipitation is performed by incubation at -20C overnight. - [Read Concentration of Oligo DNA by Ethanol Precipitation Protocol]
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]
Protocol for immunohistochemistry on fixed, paraffin-embedded sections. This method is widely used and applies to the detection of the overwhelming majority of antigens, with few exceptions for which enzymatic retrieval is required. The method uses a strong chelating agent, EDTA. Includes: Double indirect AP; AP Developing solution; Indirect immunohistochemistry with avidin-biotin and HRP; HRP Developing solution. - [Read Immunohistochemistry on Fixed, Paraffin-Embedded Sections Protocol]
Molecular and genetic toxicology studies on gene mutation in mammalian cells In vitro. Includes: G201 Mouse Lymphoma L5178Y/tk+/Cell Gene Mutation Assay; G201-R; G202 Mouse Lymphoma L5178Y/tk+/Cell Gene Mutation Screening Assay; G203 Chinese Hamster Ovary or V79 Cell Mutation Assay at the hprt Locus; G203R. - [Read Molecular and Genetic Toxicology Studies on Gene Mutation in Mammalian Cells In Vitro]
A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.
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.
Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.
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.
A single step RNA isolation protocol using Phenol Chloroform Extraction and Acid Guanidinium Thiocyanate. This RNA isolation method uses the fact that guanidinium thiocyanate can simultaneously lyse the cells and inactive cellular RNAses during the initial RNA isolation step allow a single step in the method.
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.
In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.