X-Gal Protocol (Promega Biotech)

X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) turns blue when incubated in the presence of b-glactosidase. This gene is on several of the cloning plasmids (especially, on the pUC series and lGT11 vectors). When an inserted piece of DNA is placed in the correct restriction site, the lacZ gene is interrupted and the colony does not turn the media blue (colony we want). Be sure to run controls.

1. Make 13X100 tubes with 2.5 ml LB and 0.8 agar by melting then dispensing it into the tubes and autoclaving it for only 5 min.
2. Use the tubes while hot or re-melt briefly and hold at 42 C.
3. Add 20 µl 20 mg/ml IPTG (filter sterilized in H2O), 50 µl 20 mg/ml X-gal (in Dimethylformide), and 1 µl Crb solution (100 mg/ml).
   Note: Dimethylformide seems to melt plastic so make the stock in glass or PP (or PA) oakridge tubes.
4. Add the transformed cells (try to get about 200 CFU in up to 250 µl). Vortex and overlay on a CA plate containing the appropriate antibiotic (usually Crb).
   * 2.5 ml is a little tricky to overlay neatly: do not replace cap, tilt the plate to get uniform coverage of the overlay.
5. Let the agar solidify then incubate at 37 C until the blue color develops.
6. Pick the colorless colonies to media with the correct antibiotic and verify the insert by mini plasmid preparations or colony hybridization.