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Unformatted text preview: Bioch 441 Answers to problem set #2 Young 1. The plasmid pBR322 was cut with the enzymes shown in the table below. After digestion the DNA samples were electrophoresed on an agarose gel. Molecular weight markers were also analyzed to allow you to determine the molecular weight of the DNA fragments derived from pBR322. A sample of the same pBR322 DNA was also analyzed with treatment with a restriction enzyme. Enzyme: none BamHIPst1 EcoRI SphI NcoI B+P B+E Mol. Wt. markers (kb) (-) electrode--- 6--- 5------------------ 4------ 3--------- 2------ 1 (+) electrode Draw a restriction map for pBR322 based on these data. What are the ambiguities or uncertainties? B or Pst? Pst? 1 kb E? E? 4 kb Answers to problem set #2 The band in the “none” lane represent the supercoiled plasmid DNA which migrates faster than the linear DNA of the same Kb. This is an important control because it indicates the mobility of the uncut, supercoiled DNA. Since there is a single band for cutting with Bam, Pst, Eco, and Sph, these enzymes must each cut only once, and the plasmid must be about 4 kb. Nco must not cut since the band is at the same position as in the untreated sample. Bam and Pst sites must be 1 kb apart, and Bam and Eco sites must be very close to one another since a single size fragment of about 4 kb is observed. The ambiguity is the relationship of the Bam, Pst and Eco sites. If you put the Bam site at the top of the map, is the Pst site 1 kb in the clockwise direction or in the counterclockwise direction? Ditto for the Eco site. The position of the Sph site relative to any of the others is also unknown. 2. The human genome contains about 3 X 10 9 bp (haploid genome size). If you cleave human genomic DNA with the restriction enzyme Eco RI which recognizes the sequence GAATTC what will be the average size of the fragments in the population? Assume that human DNA is random in sequence and is 50% (G+C). Approximately how many fragments of DNA will be generated? If you wanted to make a library of human genomic DNA which had a high probability of containing each and every region of the genome, about how many clones would you need? How much DNA would you need in mg? How many diploid human cells would you need to obtain this much DNA. How many sperm cells?...
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This note was uploaded on 04/15/2010 for the course BIOC 441 taught by Professor Young&parson during the Spring '08 term at University of Washington.
- Spring '08