Fragment isolation

Overview
Digestions
pGT4ΔB
Electrophoresis
DNA clean-up
Ligation
Fragment isolation
Competent cells
Transformation
Recombinants
pGTλ3758ΔH
Miniprepping
Blotting
Probe Labeling
Hybridisation
Probe Detection
PCR

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:

  1. Add 2.5 volumes of 6M Sodium Iodide to the gelslice(s)
  2. Heat to 50C to 55C to melt the gel.
  3. 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.
  4. Wash the particles several times with an ethanol containing solution (each time, collect the particles by a short centrifugation step)
  5. Elute the DNA from the particles by mixing them with water or a low salt buffer.

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DNA isolation from gel

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