Question： You have two E. coli strains, JAF1 and DAK2. JAF1 is phenotypically AmpR (Ampicillin Resistent), and DAK2 is phenotypically AmpS (Ampicillin Sensitive). You suspect that the AmpR gene is carried on a plasmid in JAF1, rather than on JAF1's chromosome, and want to design an experiment to test this hypothesis. In designing your experiment, you may assume that you have access to the followingmaterials: 1.Overnight cultures of JAF1 and DAK2 2.Competent cells of JAF1 and DAK2 a) Describe a simple experiment you could perform to test the hypothesis that the AmpR geneis carried on a plasmid in JAF1 and not on JAF1's chromosome. (Note: there is no need to describe the details of a particular procedure you might use, simply stating what you would do is sufficient-e.g. ligate A + B together). b) What specific experimental result will tell you that your hypothesis is correct? Answer： a) Here are the steps you'd want to carry out: 1. Miniprep plasmid DNA from JAF1 overnight culture 2. Transform this DNA into competent DAK2 cells 3. Plate transformed cells on LB Amp plates b) colonies on LB Amp plates Here are the best controls, and how you'd use them to interpret the data: Positive control: o transform pET into the competent DAK2 cells; plate on LB Amp plates o colonies on LB Amp plates tell you that your DAK2 cells are competent Negative control: o transform ligation buffer into competent DAK2 cells; plate on LB Amp Plates o if you don't see any colonies, confirms that your plates had Amp, that your cells were really AmpS to begin with, you had no contamination, etc. --

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