Frequently, experiments do not turn out as expected, and good scientists learn how to "troubleshoot" and test possible reasons for unexpected results. Sally's team ran this experiment but observed no colonies on the +pGLO + ampicillin + arabinose plate. List three possible reasons for this - and ways that Sally can test whether these are the cause of these experimental results.
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1. Why is ampicillin not included in the SOC medium after the cells have been heat shocked? What would happen to the cells if ampicillin were included at this step? 2. Why is uncut plasmid used as a positive control for a transformation experiment? If a researcher saw no colonies on a positive control plate, what are some possible causes? Think specifically about the transformation protocol itself. Assume the plasmid DNA was high quality and at a sufficiently high concentration that one would expect many transformants on a plate. 3. Why are there usually more colonies on the positive control plate than on the ligation plates in a typical cloning experiment?
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You see no colonies on your plate after an overnight incubation. Which of the following are possible reasons that you would see no colonies? One or more may be correct, so choose all that apply. You forgot to add ampicillin to your plate Transformation did not work and none of your bacteria took up the plasmid your EcoR1 enzyme had gone bad, so your restriction digests did not work and you accidentally transformed your bacteria with a plasmid that did not contain RFP you accidentally grabbed the wrong tube and used a plasmid that contained the kanamycin resistance gene instead of the ampicillin resistance gene
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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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