replacement Protocols

Category: PCR Protocols

DNA prepared by PCR-mediated gene disruption can be used to transform yeast in gene replacement experiments.  This protocol uses two primers, tailed with approximately 50 nucleotides homologous to the gene of interest, that target insertion of the PCR product to that locus.  Each primer ends with a universal sequence that is designed to amplify various selectable markers from plasmid templates. - [Read PCR-Mediated Gene Disruption: One-Step Method Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Many replacement vectors (e.g., the EMBL series, {lambda}2001, and {lambda}DASH) contain a series of restriction sites, arranged in opposite orientations, at each end of the central stuffer fragment. Digestion of these vectors with two different restriction enzymes yields left and right arms, a stuffer fragment, and short segments of the polycloning sites. These can easily be removed from the arms by differential precipitation with isopropanol or spun-column chromatography. - [Read Preparation of Bacteriophage lambda DNA Cleaved with Two Restriction Enzymes Protocol]

Category: Mass Spectrometry Protocols

This sample preparation method is a direct replacement for the dried droplet technique. It produces more uniform samples than the dried-droplet technique and the crystals are better adhered to the sample stage’s surface. MALDI Sample preparation. PROWL - [Read Seeded films (Vorm-Roepstorff) MALDI Protocol]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: ES Cell Growth Media and Requirements

Human embryonic stem cells are a valuable resource for research and cell replacement therapy but are notoriously cumbersome to culture. Bhatia and colleagues show that an increased dose of basic fibroblast growth factor eliminates the need for feeder laye - [Read Simplifying hESC culture]

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: Yeast Protocols: Yeast Staining Protocols

This protocol describes a method based on that of Pringle for immunofluorescent staining of yeast, as would be done in gene replacement experiments. - [Read Yeast Immunofluoresence Protocol]