Molecular Techniques

Molecular Techniques - Molecular Lab Techniques (Lecture 5,...

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Molecular Lab Techniques (Lecture 5, 8, 15, 16)
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Cloning/Recombinant DNA Methods DNA cloning = introduction of foreign DNA into bacteria or a host cell so that it will be copied when the cell replicates *but remember! Plasmids have relaxed mode of replication
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Restriction Enzymes Type II bacterial endonucleases (Naming EX: EcoRI) Recognizes a particular sequence “restriction site” and will bind to it and cleave it Restriction enzymes exist in nature to protect host DNA from foreign DNA (viruses) Host able to protect on DNA by methylating it at restriction sites to prevent restriction enzymes from binding to it
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Transformation Transformation = uptake of naked DNA Need to get the recombinant plasmid into the host cell Treat bacterial cells in mid-log phase with CaCl 2 followed by heat shock at 42°C to make the cell competent Transformation is not very efficient (1/1,000-10,000 cells) *electroporation for large plasmids
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PCR Exponential amplification of a specific DNA sequence Made possible by the discovery of Taq polymerase Reagents: dNTPs, template DNA, primer (F & R), thermostable DNA polymerase (5’ => 3’), divalent cations Steps: Denaturation ~95°C Annealing (of primers) ~50-60°C Extension ~72°C Performed in thermal cyclers Critical variables: primer design, annealing temp, [cation]
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Electrophoresis Separation of charged molecules based on migration in an electric field
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Molecular Techniques - Molecular Lab Techniques (Lecture 5,...

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