9 - Paull BIO 212 Problem Set #9 (14 pts) Due on April...

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Unformatted text preview: Paull BIO 212 Problem Set #9 (14 pts) Due on April 14/15 Handout #9: bacterial genetics cont./recombinant DNA 1. You have two bacterial strains. The first is Hfr strs met+ leu+ cys+ (where strs refers to sensitivity to the antibiotic streptomycin). The second strain is F- strr met- leu- cys- (where strr refers to resistance to the antibiotic streptomycin). met, leu, and cys are genes required for growth in the absence of methionine, leucine, and cysteine, respectively. a. (1 pt) You plate the Hfr strain on media containing methionine (met), cysteine (cys), and streptomycin. The bacteria do not grow. Why? b. (1 pt) You plate the F- strain on media containing methionine (met), cysteine (cys), and streptomycin. The bacteria do not grow. Why? c. (2 pts) You plate a mixture of the Hfr strain and the F- strain on media containing methionine (met), cysteine (cys), and streptomycin. Many colonies appear on the plate. Why? 2. The general transducing phage P1 is grown on a wild type bacterial strain (Cys+ Ura+ Trp+). The resulting phages are mixed with bacteria which require cysteine, uracil, and tryptophan (Cys Ura Trp ). Recombinants were selected on minimal medium + uracil + tryptophan (- cysteine) and 200 colonies were obtained. The 200 colonies were also analyzed on plates containing either tryptophan or uracil, as shown in the table below. Recombinant Growth on Minimal Growth on Minimal Number of Type + Trp (-Uracil -Cys) + Uracil (Trp Cys) colonies 1. - - 150 2. - + 10 3. + - 40 4. + + 0 a. (2 pts) List the genotype of each of the four possible recombinants. - b. (2 pts) What is the map order of the three genes? c. (2 pts) What experiment could be done to be certain about the order of the genes? 3. a. (1 pt) If you start out with one DNA molecule, how many would you have after 30 rounds of PCR? b. (1 pt) If you had only one primer in your PCR reaction starting with one molecule, what would you have after 30 rounds of PCR? 4. (2 pts) Shown below are the restriction sites for four endonucleases, BamHI, EcoRI, BglII, and SpeI, with the cut sites indicated with solid lines. BamH I EcoR I Imagine you have a gene that you want to clone that has BamHI sites on either end. The vector you would like to clone it into does not have a BamHI site, but does have one recognition site for each of the other enzymes, EcoRI, BglII, and SpeI. Could you clone your gene into this vector? a. Yes; you could cut the gene with BamHI and insert it into the vector by PCR. b. No, because the recognition sites in the gene and in the vector do not match. c. Yes; you could cut the gene with BamHI and insert it into the vector cut with BglII. d. No; because DNA ligase would not be able to ligate mismatched ends. e. Yes; you could cut the gene with BamHI and insert it into the vector cut with SpeI. cut ------G G ------C C cut ------G A ------C T - A T C C---T A G G---A T T C---T A A G---- Bgl II Spe I cut ------A G ------T C cut ------A C ------T G - A T C T---T A G A---- T A G T---A T C A---- ...
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