Gently flick the closed tubes with your finger to mix. Using a new sterile pipet

Question

Gently flick the closed tubes with your finger to mix. Using a new sterile pipet for each tube, pipet 100uL from each of the tubes to the corresponding plates, as shown on the diagram onto the plates. Using a new sterile loop for each plate. Spread the suspensions evenly around the surface of the agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. Stack up your plates and tape them together. Put your group name, instructor name, class section, time and day of the week on the bottom of the stack and place the stack upside down in the 37 degree C incubator until the next day. On which of the plates would you expect to find bacteria most like the original non-transformed E. coli colonies you initially observed? Explain your predictions. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions. Which plate should be compared to determine if any genetic transformation has occurred? Why? What is meant by a control plate? What purpose does a control serve?

Explanation / Answer

1. It would be in LB(-)PGLO plate. Because it does not have PGLO plasmid and shows normal growth. They are identical to the non-transformed E.coli.

2. It would be in +PGLO LB/amp and LB/amp/arab plates. Because the genetically transformed cells have taken up the PGLO plasmid which expresses the ampicillin resistance gene and these cells would survive on the plates which contain ampicillin. Moreover, the LB/amp/arab +pGLO would become responsive to UV-light and produce green florescence.

3. The LB/amp (-) PGLO and the LB/amp (+) PGLO could be compared. It is due to the cells which were not treated with DNA (-) PGLO won't express the ampicillin resistance gene and can not grow in LB/amp plates. Whereas the cells which were treated with DNA (+) PGLO must contain the PGLO plasmid, express the ampicillin resistant genes and eventually the LB/amp plate would get the transformed bacterial cells.

4. The control plate is helps to interpret experimental results. Here, both (-) PGLO plates are control plates. The (-) PGLO LB/amp control plate can be compared to LB/amp(+) PGLO plate. It shows that the transformation produces bacterial colonies which are able to grow on ampicillin. On the other hand, the comparison of (-) PGLO/LB control plate with with any of the LB/amp(+) plate shows that plasmid uptake is required for growth in the presence of ampicillin.

Overall, the (-) PGLO LB/amp plate shows no growth in this experiment and (-) PGLO LB shows normal growth and can be used as a control in this experiment.

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