We observe white colonies growing on uracil minus media after being transformed with pML104-gRNA and an HDR template. The gRNA targets a transcription factor (TF) binding site, and the HDR sequence has the targeted TF binding site deleted. What should we investigate/measure to understand why colonies are white but growing on selective media? (Select any/all that apply, and assume we are not limited by the protocols in your lab manual) a. Cas9 and gRNA expression levels b. Confirm Cas9 and gRNA (and promoters for each) are the correct sequences in the plasmid in the yeast colony c. Colony still carries the full plasmid d. Sequence the ADE2 gene of the white colony to see if mutations did occur that result in the function of ADE2 but no cutting by Cas9 + gRNA
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Let's suppose you performed the steps of gene cloning described in Figures 21.2 and $21.3 .$ 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 it 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 ampicillin-containing plates to double-check that the cells are really ampicillin resistant. e. Both $c$ and $d$ should be conducted.
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You are working in a molecular biology lab and, unbeknownst to you, your lab partner left the foreign genomic DNA that you are planning to clone on the lab bench overnight instead of storing it in the freezer. As a result, it was degraded by nucleases, but still used in the experiment. The plasmid, on the other hand, is fine. What results would you expect from your molecular cloning experiment? a. There will be no colonies on the bacterial plate. b. There will be blue colonies only. c. There will be blue and white colonies. d. The will be white colonies only.
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