When DNA is subcloned in the pUC polylinker region, β-galactosidase production is interrupted, resulting in the inability of cells to hydrolyse X-Gal. This results in the production of white colonies amongst a background of blue colonies. This experiment provides a DNA fragment together with a linear plasmid and T4 DNA ligase. Following the ligation to synthesis the recombinant plasmid, competent E. coli cells are transformed and the number of recombinant antibiotic-resistant white and blue colonies are counted. β-galactosidase activity is assayed from blue and white bacterial cells.

• For 5 lab groups
• Ligation 60 minutes; transformation 40 minutes; incubation overnight

Also required: Incubator, water bathes, automatic micropipette and tips, spectrophotometer, centrifuge and microcentrifuge.