Question

Describe the experimental procedure used to accomplish DNA transformation into bacterial cells.

Answer 1

General steps of bacterial transformation:

1. Specially prepared bacteria are mixed with DNA (e.g., from a ligation).

2. The bacteria are given a heat shock, which causes some of them to take up a plasmid.

3. 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.

4. Bacteria without a plasmid die. Each bacterium with a plasmid gives rise to a cluster of identical, plasmid-containing bacteria called a colony.

5. Several colonies are checked to identify one with the right plasmid (e.g., by PCR or restriction digest).

6. A colony containing the right plasmid is grown in bulk and used for plasmid or protein production.

The bacteria that make colonies should all contain a plasmid (which provides antibiotic resistance). However, it’s not necessarily the case that all of the plasmid-containing colonies will have the same plasmid.