MCB121 W12 L7

MCB121 W12 L7 - MCB121, C. S. Gasser MCB121 Problem Set,...

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MCB121, C. S. Gasser MCB121 Problem Set, Fundamentals of Recombinant DNA 1. As an exercise, be sure that you can follow the "pMAL Protein Fusion and Purification System" procedure on the handout from the “Molecular Methods” lecture. 2. Below are the recognition sequences of several restriction endonucleases. The sequences are written 5' to 3' and the symbol "/" indicates the cleavage site. Beside each sequence indicate which kind of end would be produced by digestion of double stranded DNA at these sites: 5'-overhang, 3'-overhang, or blunt. AGCGC/T TAC/GTA /AATT C/CATGG TT/GCAA 3. You recently isolated both a full-length cDNA clone, and a 40 kb genomic clone for a gene encoding a novel human protein called SPT. You prepare two identical southern blots, each containing several samples of human genomic DNA digested with EcoRI. Hybridization of one blot using the cDNA as a probe results in detection of a single hybridizing band in each lane. In contrast, hybridization with the entire 40 kb genomic clone results in a strong smear of hybridization encompassing all of the DNA in the lane. On the basis of what you know about the structure of the human genome, propose an explanation for the difference in these results.
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MCB121, C. S. Gasser 4. You are interested in testing the effects of changing a lysine residue to an aspartate residue in the protein encoded by the sequence below. To accomplish this you must produce an altered coding sequence in which a particular "A" residue ( boxed below) is converted to a "G" residue. You will do this using PCR-based mutagenesis. Assuming that primers of only ten bases can be effectively used for PCR, write the sequences of a set of ten base primers that would be sufficient to produce a full length double stranded copy of the entire sequence below in which the indicated "A" residue is replaced by a "G" residue. Be sure to write each primer sequence in the 5' - 3' direction (according to the primer sequence, not the gene sequence). Number your primers and mark lines above and below the sequence to show the regions to which each corresponds. One primer is already given as an example. Only the primers are requested, you need not describe their use! (there is a link on the web pages associated with this lecture that can help you with this problem!) . 1 > 1 GAATTCACAA AGATTGATAT TGATCCAAAA GCAATGGCGT ACGAGAAAGT CAACGAGCTT 61 AACCTTAAGG ACACAGAGCT ATGTCTTGGA TTACCCGGAA GAACAGAGAA GATCAAAGAA 121 GAACAAGAGG TTTCTTGCGT T AAAGTAAC AACAAGCGTC TATTTGAGGA AACTCGTGAT 181 CTTCGCAAGA TCGATCTCAA GACATACAAA AACTACCCCG AGCTTCTCAA AGCGTTAGAG 241 GACATGTTCT CTTCTTCTTG TAAGAGACTC AGAATCATGA AGGGATCCGA CGCTCCTGCT 301 CTAGACTCTT CCTTATGATC CAGAGAAGCT GAGAATCTTT TGAAGCTT Primers: 1: GAATTCACAA
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MCB121, C. S. Gasser 5. The figure below illustrates a plasmid vector. In the spaces below, provide a brief description of the utility of the indicated features/regions of the plasmid.
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This note was uploaded on 03/16/2012 for the course MCB MCB 121 taught by Professor Gasser,burgess during the Winter '11 term at UC Davis.

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MCB121 W12 L7 - MCB121, C. S. Gasser MCB121 Problem Set,...

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