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Home › Archived Educator Resources › Sanger Sequencing Archive › How Sanger Sequencing Was Done › Step 2: Insertion of Fragments into a Plasmid

Step 2: Insertion of Fragments into a Plasmid

All the small random fragments generated by shearing are inserted, or “ligated,” into a plasmid (a loop of nonessential bacterial DNA) called pUC18. The pUC18 vector allows for propagation and replication in Escherichia coli bacteria (E. coli). Each plasmid has a gene for antibiotic resistance (green in the diagram below) as well as a LacZ gene (blue) that produces blue pigment. These two genes will be important later. The fragments, shown here in various colors, are inserted in the middle of the LacZ gene.

pUC18insertion

  • How Sanger Sequencing Was Done
    • Step 1: Shearing of DNA
    • Step 2: Insertion of Fragments into a Plasmid
    • Step 3: Transformation
    • Step 4: Sub-Cloning the Sheared Fragment
    • Step 5: Colony Picking
    • Step 6: Lysing the Cell
    • Step 7: Rolling-Circle Amplification
    • Step 8: Sequencing Chemistry
    • Step 9: Post-Sequencing-Reaction Cleanup
    • Step 10: Capillary Sequencing
    • Step 11: Assembly
    • Step 12: Quality Assessment

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