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E. coli cells are transformed, then grown on agar plates containing tetracycline to select for those that have taken up plasmid.

Host transformation of E. colt cells

Host

All colonies have plasmids

Agar containing tetracycline

All colonies have plasmids

Individual colonies are transferred to matching positions on additional plates. One plate contains tetracycline the other tetracycline and ampicillin.

Colonies with recombinant plasmids colonies transferred for testing

Agar containing tetracycline colonies transferred for testing

Agar containing ampicillin + tetracycline

Agar containing tetracycline (control)

Agar containing ampicillin + tetracycline

Cells that grow on tetracycline but not on tetracycline + ampicillin contain recombinant plasmids with disrupted ampicillin resistance, hence the foreign DNA. Cells with pBR322 without foreign DNA retain ampicillin resistance and grow on both plates.

FIGURE 9-5 Use of pBR322 to clone and identify foreign DNA in E. coli. ^ Plasmid Cloning

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