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Unformatted text preview: BIO 305 Problem set for lecture 14 Winter 2008. 1. A circular plasmid is isolated and digested with Eco RI, Hind III, and Xho I, both separately and in different combinations. The fragments generated by these digestions were separated by size on an agarose gel. The results are: Eco RI HindIII Xho I 7 kb 9 kb 6 kb 2 kb 3 kb Eco RI/Hind III EcoRI/XhoI HindIII/Xho I all three 4 kb 5 kb 4 kb 3 kb 3 kb 2 kb 3 kb 2 kb 2 kb 1 kb 2 kb 1 kb Construct a map of this plasmid showing the locations and Eco RI, Hind III, and Xho I sites. 2. You are trying to generate a genomic clone of the C. elegans lin-4 gene in the plasmid vector called pUC18. You have a 2 kb PCR product that contains both lin-4 and an unknown gene (gene X). Below is a map of pUC18 and the PCR product. (a) In order to clone just the lin-14 gene, which restriction enzyme(s) should you use to digest the DNA fragment and the vector? (b) You perform your restriction digests of both the vector and the PCR fragment. Next you inactivate your restriction enzymes and perform a ligation reaction by adding DNA ligase to a mix of all your restriction fragments. Assume that you use conditions so that the ligation products have at most one inserted fragment. How many different circular plasmids that carry the Amp R gene will be present in your ligation mix? (c) You use your ligation mixture to transform a strain of E. coli that lacks both the Amp R and the lacZ genes. You plate out your transformation on plates containing X-gal. Some of your colonies are blue, some are white....
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- Winter '08