Load samples into wells and include a DNA molecular weight marker. Gel electrophoresis: prepare an agarose gel and either TAE or TBE buffer (buffer selection will depend on the duration of the run and the size of the DNA fragments).Restriction digestion: digest the DNA with a restriction enzyme, and if necessary, concentrate digested DNA.Detection methods differ based on the probe label radiolabeled probes are visualized with X-ray film or phosphorimaging, and enzymatically labeled probes are visualized with chemiluminescent substrate. The fully hybridized labeled probe molecules will remain bound to the blot. Hybridization of complementary sequences occurs during incubation, and the unhybridized probe is removed by washing with buffer. The membrane is incubated with a nucleic acid probe that has a sequence homologous to the target sequence and is labeled with radioactivity, fluorescent dye, or an enzyme capable of generating a chemiluminescent signal. Following electrophoresis, the DNA on the gel is transferred to a nylon membrane. Smaller fragments will migrate farther on the gel than larger ones. The fragments are separated by size on an agarose or polyacrylamide gel via electrophoresis.
The southern blot protocol begins with DNA extraction from the cells or tissues, which is then enzymatically digested to produce DNA fragments. Southern blots are used to determine the identity, size, and abundance of specific DNA sequences. By subscribing, you agree to receive email related to Lab Manager content and products.
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