Gel extraction
This article is missing information about other gels with other molecules.(January 2022) |
In
After DNA samples are run on an agarose gel, extraction involves four basic steps: identifying the fragments of interest, isolating the corresponding bands, isolating the DNA from those bands, and removing the accompanying salts and stain.
To begin,
Several strategies for isolating and cleaning the DNA fragment of interest exist.
Spin Column Extraction
Gel extraction kits are available from several major biotech manufacturers for a final cost of approximately 1–2 US$ per sample. Protocols included in these kits generally call for the dissolution of the gel-slice in 3 volumes of chaotropic agent at 50 °C, followed by application of the solution to a spin-column (the DNA remains in the column), a 70% ethanol wash (the DNA remains in the column, salt and impurities are washed out), and elution of the DNA in a small volume (30 µL) of water or buffer.[2]
Dialysis
The gel fragment is placed in a
Traditional
The traditional method of gel extraction involves creating a folded pocket of Parafilm wax paper and placing the agarose fragment inside. The agarose is physically compressed with a finger into a corner of the pocket, partially liquifying the gel and its contents. The liquid droplets can then be directed out of the pocket onto an exterior piece of Parafilm, where they are pipetted into a small tube. A butanol extraction removes the ethidium bromide stain, followed by a phenol/chloroform extraction of the cleaned DNA fragment.
The disadvantage of gel isolation is that background can only be removed if it can be physically identified using the UV light. If two bands are very close together, it can be hard to separate them without some contamination. In order to clearly identify the band of interest, further restriction digests may be necessary. Restriction sites unique to unwanted bands of similar size can aid in breaking up these potential contaminants.
References
- ^ Quest: An Invitrogen Publication for Discovery Vol. 4, Issue 2, pp. 44–45 (2007).
- ^ Zymoclean Gel DNA Recovery Kit Instruction Manual. http://www.zymoresearch.com/zrc/pdf/D4001i.pdf