mcb450-9s07 - Lecture 9 Recombinant DNA: Cloning and...

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Lecture 9 Recombinant DNA: Cloning and Creation of Chimeric Genes Office Hours Today (2/15/2006) 1:00-2:30, 4:30-5:30 Noyes 208 Office Hours Monday (2/19/2006) 1:00-2:30, 4:30-5:30 Noyes 208 Reviews will be scheduled for 6-8 PM on the following days Thurs (Full), Fri(Chs. 6-9), Mon (Full). CLSC B102 See web for details.
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Plasmids Small, circular, autonomously replicating DNA duplexes Frequently carry genes conferring antibiotic resistance A typical bacterial cell has one chromosome, but several hundred plasmids. Typical plasmids have cleavage sites for many restriction endonucleases.
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Plasmid Requirements Replicator: origin of replication, ori Selectable marker – gene carrying resistance to an antibiotic. Gives a means of recognizing whether a cell carries a particular plasmid. Cloning site – typically a site for a restriction endonuclease
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pBR322
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Cloning Strategy
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Construction of Chimeric Plasmids Plasmid Foreign DNA Cut plasmid and foreign DNA with the same restriction endonuclease. DNA ligase reseals the recombinant plasmid.
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Blunt End Ligase:T4 Ligase
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Adding Linkers Provides a means of adding restriction sites for DNAs that lack them
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Directional Cloning Assures that the foreign DNA will be inserted in a given direction Strategy is to cut the plasmid and the foreign DNA with two restriction endonucleases.
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This note was uploaded on 04/07/2008 for the course MCB 450 taught by Professor Mintel during the Fall '07 term at University of Illinois at Urbana–Champaign.

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mcb450-9s07 - Lecture 9 Recombinant DNA: Cloning and...

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