Clone a DNA fragment into DNA and transform bacterial phenotype.

• Complete 3 modules in 3 hours 10 min
• Mod. 1: Perform ligation of a DNA insert
• Mod. 2: Bacterial transformation/colony selection
• Mod. 3: Assay for β-galactosidase in bacterial colonies
• Perishable component refills available

When DNA is subcloned in the pUC polylinker region, ß-galactosidase production is interrupted, resulting in the inability of cells to hydrolyze 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 synthesize the recombinant plasmid, competent E. coli cells are transformed and the number of recombinant antibiotic resistant white and blue colonies are counted.

ß-galactosidase activity is assayed from blue and white bacterial cells. This experiment can be broken down into three modules: ligation, transformation, and assay of ß-galactosidase.

Additional items needed but not included: Incubation oven, two water baths, shaking incubator/waterbath, microwave/hot plate, automatic micropipet and tips, spectrophotometer, balance, centrifuge, microcentrifuge, glassware and cuvettes, distilled water, ice.