2/15/2013 background on transformation of bacteria with pGLO plasmid Experiment #5 Aim: Purpose of this lab is to have plasmid activity transformed Material: Bacteria starter plate, pGLO DNA Plasmid, microcentrifuge tubes, Ice, water bath, CaCl2 Transformation solution, (LB) agar plate, (LB/Amp) agar plate, (LB/Amp/ara) agar plate, Micropipette, and Micropipette tips. Method: Genetic transformation is a procedure which is done by taking genes from one organism and putting them in another organism.
A gene is a piece of DNA that instruct for making a new protein and from this protein organism a certain trait. A gene is inserted into an organism in order to change the organism’s trait. This procedure lab is divided into two day lab. On day one, we started the procedure with getting agar plate where HB101 bacteria were growing for 24 hours at 37C. We began by first labeling two microtubes; one with (+pGLO) and second with (-pGLO). 250ul of transformation solution which we used (CaCl2) was transfer to each tubes and placed those tubes on ice.
HB101 bacteria single colony was picked by using sterile inoculation loop and immersed into (+pGLO) tube and later immersed into (-pGLO) using same technique. Both time we used different sterile inoculation loop. The tubes were placed back into the ice after mixing well the colony each time. The pGLO plasmid DNA was added by the instructor into (+pGLO) not into (-PGLO) tube and placed the tube back into ice. The tubes were incubated on ice for 10 minutes. Once done incubating both tubes were performed heat shocks at 42 degree C temperature for 50 second.
Both tubes were immediately placed into the ice for another 2 minutes. After 2 minutes, 250ul of LB broth was added to each tube and again incubated for 10 minutes at room temperature. Once the incubation was done, we transferred 100ul of cell suspension to the plates which was provided by using the table LB/Amp| LB/Amp/ara| LB/Amp| LB| (+pGLO)| (+pGLO)| (-pGLO)| (-pGLO)| Once the cell suspension was transferred, cells were gently spread 10 swipes using inoculation loop on the agar and rotated the plate 45 degree. The plates were placed into incubator at 37 degrees by turning he tubes upside down and taping them. Result: