Blue/White Cloning of a DNA Fragment & Assay of ß-galactosidase
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.
PRODUCT UPDATE: Storage conditions for some components have changed as of 02/17/17. Be sure to download the most recent version of the literature before performing the experiment.
Group size: For 5 lab groups
Time required: Complete three modules in 3 hours 10 minutes
Kit includes: instructions, Linearized pUC plasmid & DNA fragment, T4 Ligase, BactoBeads™ for transformation, XGal in solvent, IPTG, calcium chloride, antibiotic, ReadyPour™ Luria Broth Agar, Luria broth media for recovery, growth media, assay components, plastic supplies
You need: incubation oven, two waterbaths, shaking incubator or shaking waterbath, microwave or hot plate, automatic micropipet and tips, spectrophotometer, balance, centrifuge, microcentrifuge, glassware and cuvettes, distilled water, ice.
Storage: Some Components Require Refrigerator and Freezer Storage