Biology Reference
In-Depth Information
Figure 5.4
Ethidium bromide (EtBr) can intercalate into DNA and cause the DNA to unwind. DNA
containing EtBr will fluoresce if exposed to UV irradiation. EtBr is used to visualize DNA fragments
on gels after electrophoresis. EtBR also can serve as a mutagen. Handle EtBR with great care and
protect your eyes from UV irradiation.
supercoil that has no free ends and can only unwind to a limited extent, thus
limiting the amount of EtBr that it binds. A linear DNA molecule, such as frag-
mented bacterial chromosomal DNA, can bind more EtBr and become stiffer,
extending the molecule and reducing its buoyant density.
A second method for extracting and purifying plasmid DNA exploits the
observation that within a pH range of 12.0 to 12.5, linear DNA will completely
denature, but closed circular (plasmid) DNA will not. Plasmid-containing bacteria
are treated with lysozyme to weaken the cell wall and then lysed with sodium
hydroxide and sodium dodecyl sulfate (SDS). The chromosomal DNA is dena-
tured, but upon neutralization with acidic potassium acetate, the chromosomal
DNA renatures and aggregates to form an insoluble network. The high concen-
tration of potassium acetate also causes the protein-SDS complexes and high
