How does southern blotting work

After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This appendix describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high-salt transfer buffer to promote binding of DNA to the membrane. With the high-salt buffer, the DNA becomes bound to the membrane during transfer but not permanently immobilized.

Immobilization is achieved by UV irradiation for nylon or baking for nitrocellulose. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. Transfer the denatured DNA to the membrane. Traditionally, a nitrocellulose membrane is used, although nylon or a positively charged nylon membrane may be used.

Many scientists feel nylon is better since it binds more and is less fragile. Transfer is usually done by capillary action, which takes several hours. Capillary action transfer draws the buffer up by capillary action through the gel an into the membrane, which will bind ssDNA.

You may use a vacuum blot apparatus instead of capillary action. In this procedure, a vacuum sucks SSC through the membrane. This works similarly to capillary action, except more SSC goes through the gel and membrane, so it is faster about an hour. This cross links via covalent bonds the DNA to the membrane. You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.

Probe the membrane with labeled ssDNA. This is also known as hybridization. Whatever you call it, this process relies on the ssDNA hybridizing annealing to the DNA on the membrane due to the binding of complementary strands.

A prehybridization step is required before hybridization to block non-specific sites, since you don't want your single-stranded probe binding just anywhere on the membrane. To hybridize, use the same buffer as for prehybridization, but add your specific probe. Visualize your radioactively labeled target sequence. If you used a radiolabeled 32P probe, then you would visualize by autoradiograph. Nick translation occurs and as the nick is translated down the DNA strand, the polymerase activity continues to nick while the exonuclease activity continues to fill in the nick.

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