S-Gal™: An Autoclavable Dye for Color Selection of Cloned DNA Inserts

Abstract

Blue/white selection is the standard method for detecting a cloned DNA fragment. In the absence of an insert, uninterrupted expression of the vector-encodeda-complement of β-galactosidase (β-gal), results in the hydrolysis of X-gal (5-bromo-4-chloro-3-indolyl β-d-galactoside) and the subsequent blue staining of the host colony or bacteriophage plaque expressing the carboxyterminal portion of the β-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°C is frequently required for the distinction between a positive recombinant (unstained colony or plaque) and a stained negative. S-Gal™ (3,4-cyclohexenoesculetin β-d-galactopyranoside), a novel β-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.