S-Gal™: An Autoclavable Dye for Color Selection of Cloned DNA Inserts
| Autor: | Jennifer Cosgrove, Ken Heuermann |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Viral Plaque Assay
Hot Temperature Genetic Vectors DNA Recombinant Color General Biochemistry Genetics and Molecular Biology Insert (molecular biology) law.invention Bacteriophage chemistry.chemical_compound Drug Stability law Pressure Umbelliferones Cloning Molecular Coloring Agents Incubation Chromatography High Pressure Liquid Genetics Cloning Bacteriological Techniques biology Hydrolysis Galactosides beta-Galactosidase biology.organism_classification Molecular biology Staining Solubility chemistry Recombinant DNA DNA Biotechnology |
| Zdroj: | BioTechniques. 30:1142-1147 |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/01305pf01 |
| Popis: | Blue/white selection is the standard method for detecting a cloned DNA fragment. In the absence of an insert, uninterrupted expression of the vector-encoded alpha-complement of beta-galactosidase (beta-gal), results in the hydrolysis of X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) and the subsequent blue staining of the host colony or bacteriophage plaque expressing the carboxyterminal portion of the beta-gal gene (lacZ). A white or clear colony or plaque indicates the presence of an insert. Because of its water insolubility, X-gal is dissolved in hazardous solvents such as dimethylformamide and then added to the medium following autoclaving. X-gal can be spread on previously plated medium, but this may result in an uneven color development. Also, incubation at 4 degrees C is frequently required for the distinction between a positive recombinant (unstained colony or plaque) and a stained negative. S-Gal (3,4-cyclohexenoesculetin-beta-D-galactopyranoside), a novel beta-gal substrate, is autoclavable and microwavable, allowing for dry-blending of the dye directly into the medium. Black S-Gal-stained colonies are visibly distinguishable from unstained colonies at an earlier time than X-gal. In addition, detection of the unstained signal over background is enhanced by 25% using S-Gal-containing medium, compared to medium containing X-gal. These characteristics offer convenience and better suitability for automated colony or plaque analyses. |
| Databáze: | OpenAIRE |
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