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Home › Archived Educator Resources › Sanger Sequencing Archive › How Sanger Sequencing Was Done › Step 3: Transformation

Step 3: Transformation

The plasmids are mixed into a solution containing E. coli. (E. coli normally lives in the intestines of healthy humans and other animals.) This solution is placed in a cuvette that fits into an “electroporation” machine.

A cuvette for use in the electroporation machine.

A cuvette for use in the electroporation machine.

In this machine, the plasmids are encouraged (not very gently!) to enter the bacteria cells. An electric shock is sent through the cuvette, and this opens the cell pores wide enough for the plasmids to enter. Think of it as a bacterial torture chamber ;^).

An electroporation machine induces bacteria to open their pores with an electric shock

An electroporation machine induces bacteria to open their pores with an electric shock

Some of the bacterial cells become “transformed” when they incorporate recombinant DNA. The recombinant DNA, in this case, is the 2-4-kb fragment previously sheared and ligated into the pUC18 plasmid.

transformation

  • 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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