Let's suppose you followed the protocols described in...

Let's suppose you followed the protocols described in Figures $18.2$ and 18.3. Which experiment(s) would you conduct to confirm that a white colony really contained a recombinant vector with an insert? a. Pick a white bacterial colony and restreak on plates containing X-Gal to confirm that the cells really form white colonies. b. Pick a white bacterial colony, isolate plasmid DNA, digest the plasmid DNA with a restriction enzyme, and then perform gel electrophoresis with the DNA. c. Pick a white bacterial colony and test it to see if $\beta$-galactosidase is functional within the bacterial cells. d. Pick a white bacterial colony and retest it on ampicillincontaining plates to double-check that the cells are really ampicillin resistant. e. Both $\mathrm{c}$ and $\mathrm{d}$ should be conducted.

Let's suppose you followed the protocols described in Figures $18.2$ and 18.3. Which experiment(s) would you conduct to confirm that a white colony really contained a recombinant vector with an insert?
a. Pick a white bacterial colony and restreak on plates containing X-Gal to confirm that the cells really form white colonies.
b. Pick a white bacterial colony, isolate plasmid DNA, digest the plasmid DNA with a restriction enzyme, and then perform gel electrophoresis with the DNA.
c. Pick a white bacterial colony and test it to see if $\beta$-galactosidase is functional within the bacterial cells.
d. Pick a white bacterial colony and retest it on ampicillincontaining plates to double-check that the cells are really ampicillin resistant.
e. Both $\mathrm{c}$ and $\mathrm{d}$ should be conducted.

Key Concepts

Colony Purification

Colony purification via restreaking is a standard technique to ensure that the bacterial colony is derived from a single cell and is genetically uniform. This helps to confirm that the observed phenotype is consistently due to the presence of a recombinant plasmid rather than a mixed population of cells.

Blue-White Screening

Blue-white screening is a common method in molecular cloning that exploits the ability of bacteria to produce a blue product when the lacZ gene is intact. In recombinant constructs, insertion of foreign DNA disrupts lacZ, resulting in white colonies. This technique provides a visual indicator to differentiate between colonies that likely contain an insert (white) and those that do not (blue).

Plasmid Restriction Digestion Analysis

Plasmid restriction digestion analysis involves isolating plasmid DNA from bacterial colonies and using specific restriction enzymes to cut the plasmid at known sites. The resulting fragments are then separated by gel electrophoresis. The fragment size pattern serves as a map that can confirm the presence and size of an inserted DNA fragment, thereby validating that the white colony is recombinant.

Antibiotic Selection

Antibiotic selection is employed to ensure that only bacteria harboring the plasmid of interest, which includes an antibiotic resistance gene, will grow on selective media. Although this method confirms the presence of the plasmid, it does not by itself verify that the plasmid contains the intended insert.

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