S-Gal: an autoclavable dye for color selection of cloned DNA inserts.
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.
Heuermann
K
K
Sigma-Aldrich Chemical Company, St. Louis, MO, USA. kheuermann@sial.com
Cosgrove
J
J
D001431:Bacteriological Techniques; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003116:Color; D004396:Coloring Agents; D004274:DNA, Recombinant / Q000032:analysis*; D004355:Drug Stability; D005697:Galactosides; D005822:Genetic Vectors; D006358:Hot Temperature; D006868:Hydrolysis; D011312:Pressure; D012995:Solubility; D014468:Umbelliferones; D010948:Viral Plaque Assay; D001616:beta-Galactosidase / Q000235:genetics
D004396:Coloring Agents; D004274:DNA, Recombinant; D005697:Galactosides; D014468:Umbelliferones; C101993:cyclohexenoesculetin-beta-galactoside; D001616:beta-Galactosidase