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Question 1 You have two E. coli strains, JAF1 and DAK2. JAF1 is phenotypically AmpR, and DAK2 is phenotypically AmpS. 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 overnight cultures of JAF1 and DAK2 and to competent cells of JAF1 and DAK2. a) Describe a simple experiment you could perform to test the hypothesis that the AmpR gene is 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? c) Identify one positive and one negative control you would include in your experiment above, and how you will use these controls to help you interpret your "experimental" results. 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uestion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uestion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uestion 8 You are interested in cloning the maize (corn) heat shock protein into the vector pGAL. You need to choose the restriction enzymes that you will incorporate into your PCR primers and that you will use to cut the vector (pGAL) DNA. The following are maps of the multiple cloning site of pGAL and the DNA sequence that encodes the maize heat shock protein. Another student in the lab suggests that you clone the maize gene by including an XbaI site in your forward primer and an SspI site in your reverse primer. Do you agree or disagree with this strategy? Provide two reasons for your answer.
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