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Unformatted text preview: Biology 115, Winter 2004 January 9 Problem Set #1 1. You want to express the Purple gene in bacteria. This is a mouse gene responsible for bright purple streaks commonly observed in the hair of some mouse species. You have the cloned Purple gene, with its natural promoter, and also a bacterial expression vector. Restriction maps for both of these are shown below. (A restriction map is a cartoon/map showing the relative order and position of restriction sites in a piece of DNA) Describe a strategy for cloning the Purple gene into the expression vector. This requires putting the Purple gene’s coding sequences- indicated by the boxed sequences-in the correct orientation downstream of the bacterial promoter. (Note: this process of creating a recombinant from previously cloned genes is often called subcloning ) Purple Bam HI Pst I Pst I Eco RI Bgl II Xho I Amp r ori Amp r ori Purple gene promoter Bacterial Promoter Bgl II Pst I Xho I Bam HI Eco RI Bgl II Eco RI 2. The recognition sequence for Not I is 5'-GCGGCCGC-3' 3'-CGCCGGCG-5' and recognition sequence for Eag I is 5'-CGGCCG-3'. 3'-GCCGGC-5' Both of these restriction enzymes cut their sites so as to leave 4 basepair 5' overhangs. a. Draw out both strands of the DNA sequences left by these enzymes after they have cut their sites....
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This document was uploaded on 03/14/2011.
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