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Bacterial cloning

Bacterial cloning. Especially of PCR product DNA. PCR recap. PCR gel product. Cloning. Cloning is the way in which we can take a single molecule, and make lots of bacterial cells that contain an identical molecule. These cells are clones, hence the name

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Bacterial cloning

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  1. Bacterial cloning Especially of PCR product DNA

  2. PCR recap

  3. PCR gel product

  4. Cloning • Cloning is the way in which we can take a single molecule, and make lots of bacterial cells that contain an identical molecule. • These cells are clones, hence the name • This used to be the only way to amplify DNA. It is still by far the most accurate.

  5. Plasmid vectors – circular, autonomous bacterial DNA

  6. Cloning PCR products • When we amplify DNA using PCR, it is often necessary to “clone” this DNA • We do this in order to replicate it without errors • Also, by cloning a protein coding sequence into E. coli, we can then produce the protein in the bacterium.

  7. The vector is made with a “T” overhang

  8. Taq polymerase leaves an “A” overhang • Taq is the thermostable DNA polymerase from Thermus aquaticus we used for PCR. • When Taq synthesizes a new strand, it always puts an extra “A” at the end • This can be useful, but note: other polymerases do not do this, only Taq polymerase

  9. Electroporation

  10. When the electric field is applied, • the ions move according to their • charge. • ii) Pathways are formed across the • cell membrane allowing DNA • to enter. • iii) When the electric field is • deactivated, the membrane heals.

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