1. Both the chromosome and plasmid are circular pieces of DNA in a bacterial cell. Explain, in your own words, how our plasmid isolation procedure selectively purifies plasmid DNA and avoid contaminating the plasmid sample with chromosomal DNA. (3 points)
2. When we purify the plasmid DNA, we discard the white solid and keep the liquid after the first 10 min spin. After that, we add isopropanol to the sample, mix, and then centrifuge again for 10 minutes. After this second centrifugation, we discard the liquid and keep the white solid. Explain, in your own words, why these two steps are different. (4 points)
3. Suppose that on a piece of circular plasmid, the sequence 5’GGATCC appears 4 times. This sequence is recognized by the restriction enzyme called NcoI. If we expose the plasmid DNA to the enzyme in the correct condition, how many fragments of DNA will you expect to recover? (2 points)
4. In setting up the restriction enzyme digestion reaction, the protocol states that you should add the appropriate amount of buffer. Why is adding the reaction buffer important? What property/properties of the restriction enzyme makes this essential? Explain. (3 points)
5. Refer to the following image depicting the expected outcome of our restriction digestion.
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