Gel extraction
Encyclopedia
In molecular biology
, gel extraction or gel isolation is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis. After extraction, fragments of interest can be mixed, precipitated, and enzymatically ligated together in several simple steps. This process, usually performed on plasmids, is the basis for rudimentary genetic engineering
.
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, UV light is shone on the gel in order to illuminate all the ethidium bromide
-stained DNA. Care must be taken to avoid exposing the DNA to mutagenic radiation for longer than absolutely necessary. The desired band is identified and physically removed with a cover slip
or razor blade
. The removed sliver of gel should contain the desired DNA inside. An alternative method, utilizing SYBR Safe DNA gel stain and blue-light illumination, avoids the DNA damage associated with ethidium bromide and UV light.
Several strategies for isolating and cleaning the DNA fragment of interest exist.
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.
that is permeable to fluids but impermeable to molecules at the size of DNA, thus preventing the DNA from passing through the membrane when soaked in TE buffer
. An electric field is established around the tubing (in a way similar to gel electrophresis) long enough so that the DNA is removed from the gel but remains in the tube. The tube solution can then be pipetted out and will contain the desired DNA with minimal background.
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.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, gel extraction or gel isolation is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis. After extraction, fragments of interest can be mixed, precipitated, and enzymatically ligated together in several simple steps. This process, usually performed on plasmids, is the basis for rudimentary genetic engineering
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
.
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, UV light is shone on the gel in order to illuminate all the ethidium bromide
Ethidium bromide
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...
-stained DNA. Care must be taken to avoid exposing the DNA to mutagenic radiation for longer than absolutely necessary. The desired band is identified and physically removed with a cover slip
Cover slip
A cover slip or cover glass is a thin flat piece of transparent material, usually square or rectangular, about 20 mm wide and a fraction of a millimetre thick, that is placed over objects for viewing with a microscope...
or razor blade
Razor blade
Razor blade may refer to* A razor* The Razor Blade, a 1920s racing car* Razor blade steel, a type of steel originally designed specifically for razor blades...
. The removed sliver of gel should contain the desired DNA inside. An alternative method, utilizing SYBR Safe DNA gel stain and blue-light illumination, avoids the DNA damage associated with ethidium bromide and UV light.
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.
Dialysis
The gel fragment is placed in a dialysis tubeDialysis (biochemistry)
In biochemistry, dialysis is the process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, such as dialysis tubing....
that is permeable to fluids but impermeable to molecules at the size of DNA, thus preventing the DNA from passing through the membrane when soaked in TE buffer
TE buffer
TE buffer is a commonly used buffer solution in molecular biology, especially in procedures involving DNA or RNA. "TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule that chelates cations like Mg2+...
. An electric field is established around the tubing (in a way similar to gel electrophresis) long enough so that the DNA is removed from the gel but remains in the tube. The tube solution can then be pipetted out and will contain the desired DNA with minimal background.
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.