Deck 14: Plasmid Dna Isolation and Characterization by Electrophoresis

Question 1

Explain the action of ampicillin as an inducer of plasmid replication.

Answer 1

In this problem, we are asked to explain how ampicillin functions as an inducer of plasmid replication.
Inducers start the process of gene expression or DNA replication. Plasmids are small, circular portions of DNA found in bacteria that contain useful genes not otherwise found in their chromosomal DNA.
Plasmids often contain genes that encode for antibiotic resistance - ampicillin is one such antibiotic. When a bacteria with a plasmid that contains the ampicillin resistance genes encounters ampicillin, it begins producing those proteins; it also up-regulates the replication of the plasmid so it can multiple copies of the proteins simultaneously.

Question 2

How does chloramphenicol inhibit protein synthesis?

Answer 2

In this problem, we are asked how chloramphenicol inhibits protein synthesis.
The mechanism of action for chloramphenicol involves binding to residues in both ribosomal subunits in bacteria; this binding inhibits peptidyl transferase activity, thus shutting down protein elongation in mRNA translation.

Question 3

Why is a wavelength of 600 nm used to measure growth of bacteria? Could other wavelengths be used?

Answer 3

In this problem, we are asked to explain why we use 600 nm in our spectrophotometer to determine bacterial growth and if there are any other wavelengths we could use.
600 nm is within the visible spectrum; it is used because living organisms contain many compounds that absorb light in the visible spectrum. 600 nm is in the red area of the spectrum, and many growth media are also red, orange, or yellow in color due to the components used to make them. We blank our spectrophotometer using clean media, and then check the growth of the bacteria against that blank; we must use the same wavelength in this case.

Question 4

What is the purpose of the ethanol precipitation step in the preparation of plasmids?

Question 5

Compare the isolation procedures used in this experiment with that used for chromosomal DNA (Experiment 13). Explain the differences

Question 6

Compare the two plasmid DNA isolation methods described in this experiment. Emphasize the differences.

Question 7

Why are glycerol and bromophenol blue dye added to the gel-loading buffer?

Question 8

Why is polyacrylamide electrophoresis not suitable for analysis of most plasmid DNA?

Question 9

What assumption is made about the relative electrophoretic mobility of bromophenol blue dye and plasmid DNA?

Question 10

What is the purpose of ethidium bromide in the gel electrophoresis?