All About Agarose Gel Extraction
Gel extraction is a method used to isolate a specific fragment of interest from an entire DNA through agarose gel electrophoresis. This method separates DNS fragments ranging from 50 base pair to millions using a variety of specialized apparatus. Gel extraction is a common process of genetic engineering or genetic modification. Bacteria and mice are two basic examples of genetically engineered organisms.
The agarose is only one of the three types (the other two are polyacrylamide and starch) of gels used for gel extraction because of its flexibility to separate large or tiny fragments of DNA. Once the extraction is done, scientists may mix, precipitate, or enzymitcially ligate the fragments of interest depending on the purpose of gel extraction.
How Gel Extraction is Done
There are four basic methods that scientists follow when performing agarose gel extraction.
- Identify the fragments of interest from the entire DNA structure
- Isolate the corresponding bonds
- Isolate DNA from those bands
- Remove the accompanying salts and stain from the fragments of interest
The procedure begins by illuminating the ethidium bromide- stained DNA using a UV-light with extra care to avoid exposing the sample from mutagenic radiation. Once the desired band is identified, the scientists would physically remove the gel using a razor blade. Some scientists use SYBR Safe DNA gel stain and blue-light illumination as an alternative method to avoid damages to the DNA.
Three Representative Methods of Agarose Gel Extraction
Spin Column Extraction
This method is also known as nucleic acid purification columns, a solid phase extraction method used to purify nucleic acids. Spin column extraction uses gel extraction kits that are worth $1-$2 per sample. The process begins by adding the sample to the column with the presence of chaotropic agents (i.e.sodium iodide or sodium perchlorate). Depending on the pH level and the salt content of the buffer, DNA binds to the silica, which is a solid phase, while the other molecules remain in the buffer solution. The DNA is then collected from the end of the channel walls through purification, which can be done by washing with water.
This method is also known as Visking tubing which is most preferable for semi-permeable membrane. During the process, scientists place the gel in a dialysis tube to separate molecules from the solution. Hence, the dialysis tube must be permeable only to fluids but not to materials that have the same size as DNA molecules. When soaked in a TE buffer, the DNA will not pass through the membrane. Scientists then remove the DNA from the gel by applying an electric field similar to gel electrophresis. Once done, scientists remove the solution, leaving the DNA on the tube.
This is also known as the paper slip method, wherein scientists used Parafilm wax paper to extract DNA from the gel. The process involves placing the agarose fragments inside the folded pocket of Parafilm paper. The agarose is then compressed to the corner of the pocket allowing the gel to partially liquefy. Once done, scientists pipette the droplets onto a small tube, leaving the DNA fragment inside. To purify the DNA fragment, scientists usually use butanol extraction or phenol/chloroform extraction.