VPB 321: Animal Biotechnology (2+1)

STEPS IN SOUTHERN BLOTTING

• High molecular weight DNA is digested with restriction enzymes to make it as a small fragments
• The DNA fragments are separated based on their size on an agarose gel
• A nitrocellulose membrane or nylon membrane is placed on the top of the  agraose gel in a buffer solution. A pile of tissue papers is then placed on the membrane and weight is placed above it. This makes uniform contact of gel with the membrane and facilitate the transfer of DNA fragments from the gel to membrane (blotting or transfer).
• Nitrocellulose membrane is then baked by exposing it to high temperatures (from 60 to 100 °C). Nylon membrane is exposed to UV radiation. These steps are used to ensure the permanent and covalent crosslink of the DNA present in the bands to the membranes.
• The membrane is exposed to a radiolabeled probe. This probe is a single-stranded DNA fragment which has the sequence of interest that is to be detected. This probe is incubated with the membrane and allowed to hybridize with DNA on the membrane. Probes are usually radiolabeled so that they may be detected on X-ray film, however other probes are also used which are non-radioactive such as fluorescent. After hybridization, excess unbound probe is washed away from the membrane, leaving specifically bound probe.
• The pattern of hybridization is detected by visualization on X-ray film by autoradiography in the case of a radioactive or fluorescent probes, or by development of color on the membrane if a chromogenic detection method is utilized.