33_Lieb_blotting_RFLPs

33_Lieb_blotting_RFLPs - Recombinant DNA III Blotting&...

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Unformatted text preview: Recombinant DNA III: Blotting & RFLPs J. Lieb 1 2 2 2 2 2 2 2 2 2 2 2 1 2 1 2 1 2 1 2 1 2 d d D d D d D d D d D d D d d d d d d d A Typical Expression Vector Last Lecture Vectors cDNA and Libraries grows into colony containing fragment 1 grows into colony containing fragment 2 Genomic DNA libraries partial digestion restriction map of genomic DNA (each line represents one restriction site) some fragments produced by partial digestion size selection fragments between 10 and 20 kb Creating a genomic DNA library So, now we’ve got our gene (or at least part of it) into the right vector How can I find my gene of interest in a library? Can I find other clones that contain the same gene? Are there are rearrangements in that gene in genomic DNA? Where and when is RNA from that gene produced? Where and when is Protein from that gene produced? Screening Colonies by Hybridization Some Probe sources: • cDNA from a tissue expressing the gene highly • Homologous gene • Sequence derived from the protein product of gene of interest • An earlier positive clone Other ways to find a specific clone (1) Screen an expression library with an antibody (similar to screening by hybridization) (1) Clone by complementation (1) Chromosome walking So, now we’ve got our gene (or at least part of it) into the right vector How can I find my gene in a library?...
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This note was uploaded on 06/27/2011 for the course BIOL 202 taught by Professor Kieber-hogan during the Spring '08 term at UNC.

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33_Lieb_blotting_RFLPs - Recombinant DNA III Blotting&...

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