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Molecular Cloning

Molecular Cloning. Definitions. Cloning : Obtaining a piece of DNA from its original source (Genome) and introducing it in a DNA vector Sub-cloning: Transfer a cloned DNA insert or a part thereof from one vector to another vector Plasmid recombinant:

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Molecular Cloning

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  1. Molecular Cloning

  2. Definitions • Cloning : • Obtaining a piece of DNA from its original source (Genome) and introducing it in a DNA vector • Sub-cloning: • Transfer a cloned DNA insert or a part thereof from one vector to another vector • Plasmid recombinant: • Vector into which foreign DNA was introduced • Recombinant organism • Organism with a recombinant vector

  3. Why clone? • Separate, identify, manipulate or express a specific DNA fragment 3

  4. Step 1- Separate • Two approaches: • Fragment/digest genomic DNA • Generates a vast number of fragments • May be difficult to find fragment of interest • PCR Amplification • Much less fragments • Much easier to find sequence of interest

  5. Generate compatible ends Restriction Enzyme Restriction Enzyme Fragment of interest DNA ligation Appropriate vector Intramolecular ligation Non-Recombinant Intermolecular ligation Recombinant Transformation Host Cells Recombinant cell Non-Recombinant cell Cloning DNA Recombination 1 plasmid/1 cell

  6. Plasmid Duplication 1 colony= 1 clone with 1 plasmid + 1 insert Bacterial growth Amplification of Recombinant Plasmids

  7. Screening or Identification of Recombinant Plasmid clones • Restriction mapping • Hybridization • PCR

  8. Expression • Why? • Produce the protein in a heterologous system • Produce large quantities of the protein • Purify the protein • Study the protein’s activity

  9. Amplification, Cloning & Expression of LacZ– Project II • Part I • PCR amplification & mutagenesis of LacZ • Part II • Cloning of LacZ in pUC19 • Part III • Enzyme assays of recombinant proteins

  10. Purification & Digestion of LacZAmplicon • Purification by QiaQuick • Removes buffer, salts, etc. • Removes primers • Removes enzyme • Digestion • Restriction sites NdeI and PstIwere added to amplicon sequence • Not present in LacZ sequence • Unique in pUC19 vector • Allows directional cloning

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