The DNA of prokaryotic cells (bacteria) is relatively simple in comparison to the DNA of eukaryotic cells. A bacterium has about 1/1000 as much DNA as a eukaryotic cell. In addition to chromosomal DNA, a bacterium may also carry an additional circular piece of DNA called a plasmid. Plasmids are useful to bacterial cells because they can carry genes (such as an antibiotic resistance gene) which can allow the bacteria to grow in nonideal environments.

The plasmid has proven to be a useful tool for the molecular biologist. Genes can be inserted into the plasmid which, in turn, can be incorporated into a bacterium by a process called transformation. Since the bacteria will rapidly multiply and create many copies of the gene, the molecular biologist can retrieve relatively large quantities of a protein produced by the bacteria or can use this arrangement to study the function of a particular gene.

Purpose

This unit will describe how to extract a plasmid from a bacterial cell (E .coli). This procedure is called a plasmid isolation. Isolated colonies of bacteria containing the ampicillin resistance gene (designated Ampr) inserted into the plasmid will be aseptically transferred to a bacterial growth medium, grown overnight, then chemically treated to separate bacterial chromosomal DNA and proteins from the plasmid DNA.

 

Plasmid Isolation Learner Outcomes

The following cognitive objectives should be addressed:

1. Define vocabulary related to molecular biology: plasmid, bacteria, alkali, aseptic, lyse, DNA, RNA, precipitate, and isotonic.
2. Explain or discuss the use of bacteria and plasmids in studying human genes.
3. Explain how plasmids are beneficial to bacteria.
4. Explain why scientists insert genes into plasmids.

The following laboratory objectives should be addressed. The student will be able to:

1. Use aseptic techniques in transferring bacterial colonies to bacterial growth media.
2. Pipette reagents.
3. Separate liquid (supernatant) from a pellet (precipitate) by pipetting, after centrifugation.