X-GAL (BCIG) is a widely used chromogenic substrate for β-galactosidase, which cleaves X-GAL to produce an insoluble blue compound.

Instructions:
I. Solution preparation
Mother solution preparation: Prepare X-GAL stock solution: Dissolve X-GAL in anhydrous dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) to prepare a 20 mg/mL stock solution.
Note: Protect from light, dispense in small volumes (e.g. 50–100 μL), and store at -20°C. The storage time should not be too long to avoid degradation.
II. Operation steps
1. Prepare color reaction mixture
1) For plate screening: Add X-GAL and IPTG (isopropyl-β-D-thiogalactoside) to LB agar plates. Common final concentrations: X-GAL 40 µg/mL, IPTG 0.1 mM. Add X-GAL and IPTG when the agar medium cools to about 50°C, mix well, and pour onto the plate.
2). For liquid color development: Add X-GAL and IPTG to the reaction buffer, the usual concentration is the same as for plate screening.
2. Experimental application
Blue-white screening:
1) Transform the vector containing the reporter gene (such as lacZ or lacZα) into the host bacteria (such as E. coli DH5α).
2) Spread on a plate containing X-GAL and IPTG and culture at 37°C for 12–16 hours.
3) Screening results: Blue colonies indicate positive β-gal activity (lacZ expression), and white colonies indicate negative (lacZ is not expressed).
Tissue staining:
1) After appropriate tissue fixation and permeabilization, use a reaction buffer containing X-GAL (usually PBS at pH 7.2–7.5, containing Mg²⁺).
2) Incubate at 37°C for 12–24 hours, and observe the blue precipitate under a microscope for color development.