530L_notes_lecture4

530L_notes_lecture4 - VII Making a recombinant DNA molecule...

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VII Making a recombinant DNA molecule -How do we form a covalent joined circular plasma dna (recombinant = vector + inserted dna). A. What is a ligation? 1. definintion: covalent insertion of dna fragment into a vector Watson and crick base pairing of compatible sticky ends are H-bonding. We need +DNA ligase, +rATP to covalently sealing the molecules. See ppt slide (human and corn DNA; EcoRI sites) 2. DNA Ligase: what does it do? Juxtapose 3’ hydroxyl to 5’ phosphase in DNA. B. DNA Ligase 1. Phage derived: T4 DNA ligase Derived from phage T4 DNA ligase. This ligase come from phage T4. Phage is small viruses that infect bacteria. 2. the reaction: Juxtapose 3’ hydroxyl to 5’ phosphase in DNA. 3. Potential outcomes/products a. Recombinant molecule (see ppt slide) Recombinant pet21 plasmid with inserted pho A gene. Ampicilin resistant gene, origin of replication, and MCS. b. Vector religation: what leads to this outcome? Due to incomplete digestion with one or both of our restriction endonucleases. No phoA gene inserted. **Notice both outcome are circular ddDNA plasmid that both possess ampicilin resistance gene.
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c. What is the purpose/use of the selectable marker? How do we identify the recombinant molecule if both possess the Amp resistance gene? NONTRIVIAL.
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530L_notes_lecture4 - VII Making a recombinant DNA molecule...

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