Transformation is the process by which competent bacteria take up genetic material (in the form of naked DNA) from the environment. In this lab, the process of transformation was conducted by using a plasmid containing various genes called pGLO along with the bacterium Escherichia coli. In order for transformation to occur in Escherichia coli, repeated washes in the presence of CaCl₂ followed by heat shock treatment (42°C for 50 seconds) occurred to induce the competence of the bacterium. Successful transformation results were expressed by phenotypic characteristics such as growth in the medium and glowing from exposure of UV light from the pGLO plasmid genes (β-lactamase gene and green fluorescent protein gene).
Introduction Genetic transformation …show more content…
coli, 100Mm CaCl₂, and six Luria-Bertani medium plates. First, two micro-centrifuge tubes were obtained and labeled pGLO+ and pGLO-. Using a micropipette, 250 μL of E. coli HB101 mixed in CaCl₂ buffer was transferred into each tube, and the two tubes were placed in an ice bucket. In addition, 8 μL of the pGLO plasmid was added to the pGLO+ tube, and the tube was placed back into the ice bucket for ten minutes. While the tubes were in the ice bucket, six Luria- Bertani plates were obtained and labeled LB pGLO+, LB pGLO-, LB/amp pGLO+, LB/amp pGLO-, LB/amp/ara pGLO+, and LB/amp/ara pGLO-. Once the ten minutes past, both of the tubes were placed in the heating block (42°C for 50 seconds). Immediately after the heating block, both of the tubes were placed back into the ice bath for 10 min. Next, 250 μL of fresh LB broth was aseptically transferred to both tubes, and the tubes were placed in the heating block (37°C for 10 minutes). Finally, each transformation plate was prepared by pipetting 100 μL of cells from each tube to its corresponding label on each plate. Using an ethanol sterilized glass spreader, the 100 μL of cells was spread evenly around the surface of each plate. Lastly, the six plates were tapped together and placed in the incubator (37°C for 24 hours). After incubation, a hand-held UV lamp was used to observe the gfp expression along with bacterial
Introduction Genetic transformation …show more content…
coli, 100Mm CaCl₂, and six Luria-Bertani medium plates. First, two micro-centrifuge tubes were obtained and labeled pGLO+ and pGLO-. Using a micropipette, 250 μL of E. coli HB101 mixed in CaCl₂ buffer was transferred into each tube, and the two tubes were placed in an ice bucket. In addition, 8 μL of the pGLO plasmid was added to the pGLO+ tube, and the tube was placed back into the ice bucket for ten minutes. While the tubes were in the ice bucket, six Luria- Bertani plates were obtained and labeled LB pGLO+, LB pGLO-, LB/amp pGLO+, LB/amp pGLO-, LB/amp/ara pGLO+, and LB/amp/ara pGLO-. Once the ten minutes past, both of the tubes were placed in the heating block (42°C for 50 seconds). Immediately after the heating block, both of the tubes were placed back into the ice bath for 10 min. Next, 250 μL of fresh LB broth was aseptically transferred to both tubes, and the tubes were placed in the heating block (37°C for 10 minutes). Finally, each transformation plate was prepared by pipetting 100 μL of cells from each tube to its corresponding label on each plate. Using an ethanol sterilized glass spreader, the 100 μL of cells was spread evenly around the surface of each plate. Lastly, the six plates were tapped together and placed in the incubator (37°C for 24 hours). After incubation, a hand-held UV lamp was used to observe the gfp expression along with bacterial