mcb450-9s09

mcb450-9s09 - Lecture 9 Recombinant DNA: Cloning and...

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Lecture 9 Recombinant DNA: Cloning and Creation of Chimeric Genes Office Hours Noyes 208 Today (2/17/09), 1:30-3:00 Hour Examination Thursday, 2/19/09 Chs. 1-9 Bring a calculator.
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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 This enzyme can be used in the laboratory to add DNA segments contain a sequence conferring susceptibility to restriction endonucleases as shown on the next slide.
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Adding Linkers Provides a means of adding restriction sites for DNAs that lack them T4 ligase, a blunt end ligase.
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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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mcb450-9s09 - Lecture 9 Recombinant DNA: Cloning and...

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