In a control experiment, a plasmid containing a HindIII recognition sequence within a kanamycin resistance gene is cut with HindIII, re-ligated, and used to transform E. coli K12 cells. Kanamycin-resistant colonies are selected, and plasmid DNA from these colonies is subjected to electrophoresis. Most of the colonies contain plasmids that produce single bands that migrate at the same rate as the original intact plasmid. A few colonies, however, produce two bands, one of original size and one that migrates much less far down the gel. Diagram the origin of this slow band as a product of ligation.

Key Concepts

Plasmid Structure and Function

Plasmids are small, circular DNA molecules found in bacteria that can replicate independently of chromosomal DNA. They often carry genes that confer advantageous traits, such as antibiotic resistance. In this experiment, the plasmid contains a kanamycin resistance gene, which allows transformed E. coli to survive in the presence of the antibiotic, making it a useful tool for genetic engineering.

Restriction Enzymes and Ligation

Restriction enzymes, like HindIII, are proteins that cut DNA at specific sequences, creating fragments with 'sticky' or 'blunt' ends. After cutting, these fragments can be rejoined through a process called ligation, where DNA ligase facilitates the formation of phosphodiester bonds between the DNA ends. This is crucial for inserting new genetic material into plasmids, as seen in the experiment.

Gel Electrophoresis

Gel electrophoresis is a technique used to separate DNA fragments based on their size. When an electric current is applied, smaller fragments migrate faster through the gel matrix than larger ones. In this experiment, the presence of two bands in some colonies indicates that the plasmids have undergone successful ligation, resulting in a larger fragment that migrates more slowly compared to the original plasmid.