X-β-Gal, 2.5 g

Ideal for colorimetric detection of β-galactosidase activity, for instance during blue-/white selection.

Being a galacto pyranoside, IPTG inhibits the lac repressor, therefore leading to the induction the lac promotor. Subsequently, using the plasmid as matrix the C-terminal 146 amino acid fragment of the bacterial β-galactosidase is expressed, which functions as so called α-donor and complements the C-terminally deleted bacterial β-galactosidase. The then functional β-galactosidase restricts the galactoside X-Gal, resulting in a blue-coloured dye - therefore, these lac+ clones are coloured dark blue.
Since the C-terminal part of the α-donor is located upstream and the N-terminal part is located downstream of the multi cloning site of the plasmid, inserted DNA normally leads to frame shift or at least to major chain elongation, resulting in an unfunctional α-donor and no functional galactosidase - recombinant clones are white. The appearance of light blue clones either results form very short insertions or from the fact that in some plasmids the C-terminal fragment cloned upstream of the MCS is sufficient for α-complementation and restriction of a small amount of X-Gal.

Working solution: for blue/white selection of recombinant clones on agar plates use 0.3 μl stock solution per ml broth/agar.