Molecular Methods Probs

Molecular Methods Probs - MCB121, C. S. Gasser MCB121...

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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
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Molecular Methods Probs - MCB121, C. S. Gasser MCB121...

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