A Southern blot is a method in molecular biology of enhancing the result of an agarose gel electrophoresis by marking specific DNA sequences. The method is named after its inventor, the British biologist Edwin Southern. This caused other blot methods to be named similarly as plays on Southern's name (for example, Western blot, Northern blot, Southwestern blot).
Method:
1. Restriction endonucleases are used to break DNA strands into fragments. 2. The fragments are then electrophoresed on a gel to separate cut DNA based on size. 3. If DNA is larger than 15 kb, prior to blotting, the gel may be treated with a weak acid, such as dilute HCl, which acts to depurinate the DNA fragments. This breaks the DNA into smaller pieces that will be able to complete the transfer more efficiently than larger fragments. 4. The gel from the DNA electrophoresis is treated with an alkaline solution (typically containing sodium hydroxide) to cause the double-stranded DNA to denature, separating it into single strands. Denaturation is necessary so that the DNA will stick to the membrane and be hybridized by the probe (see below). 5. A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel). This causes the DNA to move from the gel onto the membrane by capillary action, where it sticks. 6. The membrane is then baked (in the case of nitrocellulose) or exposed to ultraviolet radiation (nylon) to permanently crosslink the DNA to the membrane. 7. The membrane is now treated with a hybridization probe - an isolated DNA molecule with a specific sequence that pairs with the appropriate sequence. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA.
1 8. After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualized on x-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane itself if a chromogenic detection is used.
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