DNA fragment isolation from gel
For several purposes it can be necessary to purify DNA fragments from
from bands in an agarose gel.
Chaotropic salts have the property to destroy the secondary
structure of polymers. Consequently, they are not only strong
protein-denaturing agents, but they will also solubilise an agarose
gel at moderate temperatures.
In general, DNA binds to silica (glass) in high concentrations of
chaotropic salt (like Sodium Iodide NaI) and elutes in low salt. The
mechanism of DNA binding to silica in high salt has not been
described, but may involve chaotropic salt disruption of the water
structure around negatively charged silica, allowing a cation bridge to
form between it and the negatively charged phosphate backbone of DNA.
When the salt is removed, rehydration of the silica matrix breaks the
attraction between the matrix and DNA. The fact that DNA binds in high
salt and elutes in low salt makes this method especially useful as a
purification procedure. Since the DNA is eluted with either water or a
low salt buffer, it can be used immediately in subsequent reactions
without precipitation or other further manipulation.
In general, the steps in the isolation of DNA fragments from
agarose gel slices are:
- Add 2.5 volumes of 6M Sodium Iodide to the gelslice(s)
- Heat to 50°C to 55°C to melt the gel.
- Add a small volume of a suspension of glass particles (or
so-called Diatomaceous Earth), and rock for about 30 minutes, to
bind the DNA to the particles.
- Wash the particles several times with an ethanol containing
solution (each time, collect the particles by a short centrifugation
- Elute the DNA from the particles by mixing them with water or a
low salt buffer.