S-Gal (TM): An autoclavable dye for color selection of cloned DNA inserts

Blue/White selection is the standard method for detecting a cloned DNA frag ment. In the absence of an insert, uninterrupted expression of the vector-e ncoded alpha -complement of beta -galactosidase (beta -gal), results in the hydrolysis of X-gal (5-bromo-4-chloro-3-indolyl-beta -D-galactoside) and t he subsequent blue staining of the host colony or bacteriophage plaque expr essing 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 dimet hylformamide and then added to the medium following autoclaving. X-gal can be spread on previously plated medium, but this may result in an uneven col or development. Also, incubation at 4 degreesC is frequently required for t he distinction between a positive recombinant (unstained colony or plaque) and a stained negative. S-Gal(TM) (3,4-cyclohexenoesculetin-beta -D-galacto pyranoside), 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 ov er background is enhanced by 25% using S-Gal-containing medium, compared to medium containing X-gal. These characteristics offer convenience and bette r suitability for automated colony or plaque analyses.