Investigating CSI – Background material Table of Contents I ...

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 radioactively-labeled 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 below 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.
To use this method to quantify DNA it is necessary to compare your results to a
standard curve. You would need to use a smaple that contains a known quantity
of DNA.
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