Southern Blot

From the Cornell University site:

http://drylab1.vet.cornell.edu/Block4/Reference/Diagnostics/south.html

Southern Blot

The Southern blot is used to detect the presence of a particular bit of DNA in a sample. The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.

DNA is extracted from the cells and purified

Restriction Digest
DNA is restricted (cut) with enzymes.

DNA fragments
In this example, a large piece of DNA is chopped into smaller pieces using a restriction enzyme.

Loading the Gel
The DNA is loaded into a well of the gel matrix.

Lane 1 contains size standards (a mix of known DNA fragments)
Lane 2 contains the restricted DNA
Lane 3 contains unrestricted (whole) DNA

Running the Gel
An electric current is passed through the gel and the DNA moves away from the negative electrode. The distance moved depends on the size of the DNA fragment. Standards are used to quantitate the size. Unrestricted, large DNA runs as a smear due to random shearing (breaking) of the DNA. The DNA can be visualized by staining first with a fluorescent dye and then lighting with UV.

Transfer of the DNA to a Membrane
The DNA is first denatured (made single stranded - usually by raising the pH) and then transferred out of the gel and onto a membrane. The transfer can be done electrically or by capillary action with a high salt solution.

Development of the blot
A labelled probe specific for the gene in question is incubated with the blot. The blot is washed to remove non-specifically bound probe and then a development step allows visualization of the DNA that is bound. See Southern Up Close for a detailed description of this process. In this example the gene is found in the second largest fragment of the restricted DNA. In the unrestricted DNA it migrates more slowly because it is part of a larger molecule.

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DNA is extracted from the cells and purified
Restriction Digest DNA is restricted (cut) with enzymes.
DNA fragments In this example, a large piece of DNA is chopped into smaller pieces using a restriction enzyme.
Loading the Gel The DNA is loaded into a well of the gel matrix. Lane 1 contains size standards (a mix of known DNA fragments) Lane 2 contains the restricted DNA Lane 3 contains unrestricted (whole) DNA
Running the Gel An electric current is passed through the gel and the DNA moves away from the negative electrode. The distance moved depends on the size of the DNA fragment. Standards are used to quantitate the size. Unrestricted, large DNA runs as a smear due to random shearing (breaking) of the DNA. The DNA can be visualized by staining first with a fluorescent dye and then lighting with UV.
Transfer of the DNA to a Membrane The DNA is first denatured (made single stranded - usually by raising the pH) and then transferred out of the gel and onto a membrane. The transfer can be done electrically or by capillary action with a high salt solution.
Development of the blot A labelled probe specific for the gene in question is incubated with the blot. The blot is washed to remove non-specifically bound probe and then a development step allows visualization of the DNA that is bound. See Southern Up Close for a detailed description of this process. In this example the gene is found in the second largest fragment of the restricted DNA. In the unrestricted DNA it migrates more slowly because it is part of a larger molecule.