The materials used in the transformation of Escherichia Coli include a transformation solution, a starter plate of bacteria colonies, and pGLO. A bacterial suspension is created by transferring 250 μl of transformation into two sterile 2.0 microcentrifuge tubes of different colors. One of the tubes is labeled +pGLO and the other is labeled –pGLO. Once 250 μl transformation solution is in each of the tubes they are placed on a round floating tube rack and then placed on ice. To complete the bacterial suspension, 2 large or 3 small colonies of bacteria are taken from the starter plate using a sterile loop. Holding the +pGLO tube between two fingers, the loop with the bacterial colonies is placed in the transformation solution. To ensure that the entire colony is completely dispersed in the transformation solution the loop is spun vigorously until there are no floating chunks remaining in the solution. Using a new sterile loop, this same process is …show more content…
To mix the tubes and re-suspend the bacterial cells the tubes are gently flicked. Using a fresh pipette, the cell suspension is transferred onto the labeled culture plates as such: 200μl of the –PGLO onto the LB plate, 200μl of the –pGLO onto the LB/Amp plate, 200μl of +pGLO onto the LB/Amp plate, and 200μl of the +pGLO onto the LB/Amp/ara plate. Using a new sterile disposable L-shaped spreader for each plate, the suspension is spread evenly around the agar by skating the straight edge of the spreader around the entire plate surface. To keep from contaminating the plates, the lid should only be opened wide. Once the bacterial cells are spread on each plate the plates sit for five minutes at room temperature so that the liquid suspension can be soaked up by the culture medium. The plates should then be rubber banded together and placed upside down in the designated collection tray. This concludes the first week of the