1)How can you indicate that the bacteria have taken up the plasmid?
2)What is the purpose of the LB/amp(-) plate? Should there be bacterial growth on the plate?
3) Why would one compare plates LB/amp (-) and LB/amp (+)?
Suppose y varies directly with x. If y = 6 when x = -2, find x when y = 15.
a. ⅕
b. 5
c. -5
d. -⅕
1) To indicate that bacteria have taken up the plasmid, you can use a process called plasmid transformation. This involves introducing the plasmid into the bacteria and selecting for those bacteria that have successfully incorporated the plasmid into their genomes. Here's how you can indicate that the bacteria have taken up the plasmid:
a) First, prepare a LB/amp (+) agar plate by adding the antibiotic ampicillin to Luria-Bertani (LB) agar. Ampicillin is normally used as a selective marker, as only bacteria with the plasmid containing the ampicillin resistance gene will be able to grow on this plate.
b) Transfer the transformed bacteria onto the LB/amp (+) agar plate using a sterile inoculation loop or spreader. Make sure to streak the bacteria evenly across the plate.
c) Incubate the plate at the appropriate temperature (usually 37°C for E. coli) for a sufficient period, usually overnight.
d) After incubation, observe the plate for bacterial growth. If colonies appear on the plate, it indicates that the bacteria have successfully taken up the plasmid and possess the ampicillin resistance gene.
2) The LB/amp (-) plate is a control plate used to determine if the antibiotic ampicillin itself inhibits bacterial growth. The purpose of this plate is to ensure that the bacteria used in the experiment are not naturally resistant to ampicillin, which would interfere with the transformation process.
Ideally, there should be no bacterial growth on the LB/amp (-) plate. If there is growth, it suggests that either the transformed bacteria were not properly prepared or there might be contamination in the transformation process. In such cases, it becomes challenging to determine if the observed bacterial growth on the LB/amp (+) plate is due to successful transformation or preexisting resistance to ampicillin.
3) Comparing LB/amp (-) and LB/amp (+) plates allows for the differentiation between bacteria that have taken up the plasmid (transformed bacteria) and those that have not. Here's why you would compare these plates:
a) The LB/amp (-) plate serves as a negative control. It helps identify any bacterial colonies that grow despite the absence of ampicillin in the selective medium. Unexpected growth on this plate could indicate contamination or the presence of naturally occurring ampicillin-resistant bacteria.
b) The LB/amp (+) plate, on the other hand, contains ampicillin, allowing only transformed bacteria to grow. These transformed bacteria should possess the plasmid with the ampicillin resistance gene. By comparing the growth on this plate to the LB/amp (-) plate, you can determine if the observed growth is due to the successful uptake of the plasmid or other factors like contamination or preexisting resistance.
In summary, the comparison of LB/amp (-) and LB/amp (+) plates helps ensure the specificity of the ampicillin resistance gene as a marker for successful plasmid uptake and helps verify the success of the transformation process.