Blue/White Color Screening lacZ lacZ insert functional enzyme nonfunctional enzyme X-gal product X-gal product 33. It relies on the activity of β-galactosidase, an enzyme occurring in E. coli, which cleaves lactose into glucose and galactose. Blue-white screening of bacterial colonies is a popular and effective molecular biology tool often used to detect recombinant bacteria in cloning experiments. Pour plates and allow to cool to room temperature (usually at least 30 minutes) before use. Note: Blue/White Selection plates are generally stable for … Spread transformed competent cells as desired. 5. Add screening antibiotic of choice (Ampicillin, Kanamycin, Carbenicillin, etc). Selecting Colonies with Recombinant Plasmids Blue/White Screening of Bacterial Colonies X-Gal/IPTG Plates Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria. Central to this technique is the enzymatic activity of β-galactosidase, a tetrameric enzyme encoded by the lacZ α gene in E. Academia.edu is a platform for academics to share research papers. Blue/White Cloning of a DNA Fragment and EDVO-Kit # 300 3 Assay of b-Galactosidase Storage Reagents for DNA Ligation L1 DNA Vector Linearized with Eco RI and DNA Fragments -20° C Freezer L2 Control Superhelical Plasmid -20° C Freezer L3 T4 DNA Ligase/ATP/Buffer Reaction Tube -20° C Freezer L4 TE Buffer, Sterile Room Temp. 6. Identification of Recombinant Bacteria. 4. In the simple cloning system we have used so far, we used antibiotic resistance to select the desired clones. Blue-white screening provides a convenient and powerful way to distinguish bacterial colonies or phage plaques that contain a cloning vector with a DNA insert, from those containing empty vectors with no insert DNA. Bio 181: Blue/White screening (pBLU) A central problem of cloning is the identification of a desired clone among countless bacteria, transformed & untransformed. We also acknowledge previous National Science Foundation support under grant numbers 1246120, 1525057, … The LibreTexts libraries are Powered by MindTouch ® and are supported by the Department of Education Open Textbook Pilot Project, the UC Davis Office of the Provost, the UC Davis Library, the California State University Affordable Learning Solutions Program, and Merlot. The method is based on the blue pigment that forms when beta-galactosidase catalyzes hydrolysis of the synthetic substrate X-gal.