• Spread the following on a LB (+appropriate antibiotic) plate (If you are going to plate 100ul of transformed cells): 100ul of 100mM IPTG + 40ul of X-gal (20mg/ml).

• Perform transformation of ligation into the bacteria (note: make sure you are using the right host bacterial strain for the blue white selection, also see the background section below) and then spread them on the X-gal containing plates. 
• After overnight incubation, store the plate in 4 ⁰ C for several hours. This allows the blue color to develop fully. Colonies that contain active b -galactosidase are pale blue in center and dense blue at their periphery. White colonies occassionally show a faint blue spot in the center, but these are colorless at the periphery.

X-gal-X-gal is very expensive, and hence avoid wastage. X-gal stock solution can be prepared by dissolving 100mg of X-gal in 5ml of dimethyl formamide. X-gal is light sensitive and hence store in a glass or polypropylene containers wrapped with aluminium foil. Stock sloution need to be store in -20 ⁰ C

IPTG (Isopropyl Thio- b -D-Galactoside, F.W.= 238.31)-To get a 100mM stock solution, dissolve 0.238g in 8ml of dH2O and then bring the volume to 10ml with dH2O. Filter sterilize the solution by passing it through 0.22 micron filter, prepare 1ml aliquots and store them in -20 ⁰ C.

Background: Many vectors (such as pUC series) carry coding information for the first 146 amino acids of the b -galactosidase gene. Embedded in this coding region is the polycloning site (does not disrupt the reading frame of the gene) into which insert DNA is cloned. When expressed, this 146 amino acid fragment of b -galactosidase protein is incapable of acting on the chromogenic substrate (X-gal). But when expressed in appropriate host cells which expresses the carboxyl terminal fragment of the b -galactosidase protein, these two protein fragments can associate to form an enzymatically active protein. This is called as a -complementation and such cells turn blue when plated on plates containing X-gal. But if the insert DNA has gotten cloned in the polycloning site, it almost invariably results in production of an aminoterminal fragment that is not capable of a -complementation and hence those colonies remain white. Ref: Ullmann et al, 1967, J. Mol.Biol. 24:339

** Note: Need strain with an IPTG inducible promoter; e.g. XL 1Blue,DH5a ,Top10F’…