Beyond the Identification of Transcribed Sequences:
Functional, Evolutionary and Expression Analysis
12th International Workshop
October 25-28, 2002
Washington, DC


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S-Gal: An Autoclavable, Water-Soluble Dye for Enhanced Color-Selection of Cloned DNA Inserts

Sara Borchardt and Ken Heuermann
Sigma-Aldrich Biotechnology, St. Louis, MO
Telephone: 314-289-8496, x8610
Fax: 314-286-7645
Email: kheuermann@sial.com

S-Gal (3,4-cyclohexenoesculetin-beta-D-galactopyranoside) outperforms X-gal as a beta-galactosidase substrate for automated and non-automated molecular cloning applications involving color selection.  Insertion of a DNA fragment into a vector multiple cloning region embedded in the alpha-complement of the lacZ gene disrupts beta-galactosidase activity in the host, resulting in the formation of a colorless or cream-colored colony.  Dark black staining of a colony indicates uninterrupted expression of the alpha-complement (no insertion). Better contrast is observed between the stained and unstained colonies and the background of the plated medium when using S-Gal as opposed to X-gal. As a result, colonies representing recombinants can be distinguished from those containing the parental vector at an earlier timepoint, typically between 19 to 22 hours following plating, using pUC18 and DH5alpha host strain. Other standard E. coli strains (JM109, XL-1Blue, and NovaBlue) have been successfully tested using S-Gal. Additionally, pSTBlue-1 transformants produce black colonies in the presence of kanamycin. 

Autoclavable/microwavable S-Gal is blended in LB agar (with IPTG <isopropyl beta-D-thiogalactoside> and ferric ammonium citrate) at a final plated concentration of 300 micrograms per milliliter.  Kanamycin can be added to this blend prior to autoclaving or microwaving for antibiotic selection. Addition of ampicillin to the blend confers selection to microwaved medium.  A water-soluble S-Gal sodium salt derivative has been developed which allows for customized adjustment of dye concentration and addition of S-Gal to alternative medium formulations. As in case of the free base form found in the blended agar preparation, the sodium salt has been shown to be heat-stable. Both are light-stable (X-gal is not) and have been tested to be stable in prepared medium containing 100 micrograms per milliliter of ampicillin for up to one month, with no effect on color development or antibiotic selection.



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