Discussion 9 key Fall 2007

Discussion 9 key Fall 2007 - Amp R gene Pst I When inserted...

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GENETICS (BIO 325) Fall 2007 Discussion sheet 9 key _____________________________________________________________________________ _ 1. Bam H1 and Bgl II; 5'-GATC-3' 2. Some cohesive ends can form perfect Watson-Crick pairs, and therefore can be ligated; others cannot. Ligations (1) and (2), each involving two ends created by a single restriction enzyme ( Bam H1 and Xba I, respectively), regenerate a restriction site for the corresponding enzyme. Ligation (4) is successful because the cohesive ends are compatible, but ligation creates a "hybrid" hexanucleotide sequence that is not a restriction site for either Xba I or Nhe I. Ligations (3) and (5) cannot occur. 3. (a) 5'-AT-3' 5'-CG AT-3' Overhangs are underlined. 3'-TAGC -5' TA-5' (b) 4 (c) Cla I: cannot be determined Taq I: yes 4. 5. (a) Bam H1: one 4 kb; Bgl I: three 0.3, 1.5, 2.2 kb; Eco RI: one 4 kb; Pst I: one 4 kb (b) Two 0.7, 3.3 kb (c) You would choose an enzyme with a single restriction site in pBR322 within the Tet R gene ( Bam H1 or Sal I) or within the
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Unformatted text preview: Amp R gene ( Pst I). When inserted within a plasmid gene, the foreign DNA abolishes expression of the gene. Specifically, if inserted within Tet R or Amp R , sensitivity to or ampicillin would result. (d) You would select an E. coli strain carrying a "hybrid" plasmid (foreign DNA inserted into pBR322) by its pattern of sensitivity or resistance to or ampicillin. For example, if the foreign DNA were inserted into the Bam H1 site within the Tet R gene, you would select for ampicillin resistance and sensitivity. Resistance to ampicillin selects for the presence of the plasmid. Sensitivity to selects for the foreign DNA insert. 6. (a) The medium should contain ampicillin, X-gal, and nutrients. (b) Blue; the β-gal gene is functional. (c) White; the β-gal gene is disrupted by the insertion of the 1.0-kb Xba I fragment, and no β-galactosidase protein is synthesized. (d) 1 7. 5'-TTGCA-3' 2...
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Discussion 9 key Fall 2007 - Amp R gene Pst I When inserted...

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