Question

Explain the various steps involved in bacterial transformation.

Answer 1

Transformation and selection of bacteria are key steps in DNA cloning. ... After a ligation, the next step is to transfer the DNA into bacteria in a process called transformation. Then, we can use antibiotic selection and DNA analysis methods to identify bacteria that contain the plasmid we're looking for.

Answer 2

• Steps of bacterial transformation and selection
• Specially prepared bacteria are mixed with DNA (e.g., from a ligation).

• The bacteria are given a heat shock, which causes some of them to take up a plasmid. [How does that work?]

• Plasmids used in cloning contain an antibiotic resistance gene. Thus, all of the bacteria are placed on an antibiotic plate to select for ones that took up a plasmid.

• Diagram of a plasmid. The plasmid contains an antibiotic resistance gene, a promoter to drive gene expression in bacteria, and the target gene inserted during the ligation.
• Bacteria without a plasmid die. Each bacterium with a plasmid gives rise to a cluster of identical, plasmid-containing bacteria called a colony.
• Several colonies are checked to identify one with the right plasmid (e.g., by PCR or restriction digest).
• A colony containing the right plasmid is grown in bulk and used for plasmid or protein production.