What are the steps involved in southern blotting?

The steps involved in southern blotting are described in numerical order below. 

1. DNA digestion: Cut the DNA using the correct restriction enzyme to cleave the DNA molecule at precise sequences known as recognition sites. 
2. Gel Electrophoresis: Separate the DNA fragments by running them through an agarose gel; acrylamide gels offer an alternative option for effectively separating smaller DNA fragments (typically those less than 800 base pairs in length). 
3. Blotting: The DNA is then transferred to a nylon membrane that has a positive charge. This transfer process typically involves upward-transfer through capillary action. In this process, liquid moves upward against gravity to transfer the DNA fragments from the gel to the membrane, where they bind to the ssDNA. 
4. Probe labeling: A nucleic acid probe (which shares a similar sequence to the target DNA being studied) is tagged with a marker such as radioactivity,  an enzyme, or a fluorescent dye. The marker emits a signal, (e.g. light) when it interacts with the target sequence, enabling the detection of the specific DNA fragment. 
5. Hybridization and washing: The labeled probe is mixed with the immobilized DNA fragments on the blot and incubated under conditions which favor the binding of complementary sequences. This process involves both prehybridization and hybridization itself. 
1. Following hybridization, any unbound probe is eliminated through a series of buffer washes. Low-stringency washes (e.g. solutions like 2X SSC) remove both the hybridization solution and any unbound probe. On the other hand, high-stringency washes (e.g. 0.1X SSC) specifically target partially hybridized probe molecules, ensuring that only fully hybridized probes remain bound to their complementary sequences on the blot.
6. Detection: The labeled probe that has bound to its target sequences on the blot is identified using the appropriate technique based on the specific label utilized. For instance, enzymatically labeled probes are usually detected by treating the blot with a chemiluminescent substrate and then captured by X-ray film for visualization.