Agarose gel electrophoresis is a method of choice for the identification, purification, and separation of the DNA fragments. DNA fragments from the gel are routinely extracted for various downstream processing. These include, cloning, radio-labeling, in vitro transcription, microinjection and sequencing of the DNA molecules.
Gel extraction of DNA fragments is mainly done to remove proteins and salts that incorporate from certain reactions. Therefore, in order to use the DNA fragments for downstream processing, these components musts be removed. For example, a PCR amplification or restriction enzyme digestion reaction contains factors which inhibit further applications of the DNA fragment.
There are various methods employed for the extraction of DNA fragments from agarose gel. Among the methods used, silica-membrane containing spin-column based DNA extraction is the most widely used. This is the quickest method to effectively purify DNA fragments from agarose gels.
However, DNA extraction using this method is not always efficient and depends on following factors:
1) Size of the DNA fragment
The first factor affecting the extraction of DNA fragment from the agaorose gel is the size of the DNA fragment itself. It has been observed that DNA fragments of size 500-5000bp are extracted most efficiently from the agarose gels. However, DNA fragments smaller than 500bp or lager than 5000bp, are poorly recovered from the gel.
2) Concentration of agarose in the gel
Concentration of agarose in the gel has a significant effect on the extraction of DNA fragments. It has been observed that recovery of DNA fragments is inversely proportional to the concentration of agarose in the gel. As the concentration of agarose in the gel increases, recovery of DNA molecules decreases. Therefore, in order to carryout good recovery of DNA fragments, less concentration of agarose is used in the gel.
Generally, for gel extraction, 0.7-1% agarose gels are prepared. However, if the size of the DNA fragment is less than 300bp you will have to use higher concentration of agarose (1.2-1.5%) in the gel. We routinely use 0.7% agarose gels for extracting DNA molecules.
3) Voltage applied to the gel
The applied voltage also significantly affects the extraction of DNA fragments. In order to extract any DNA molecule from the gel you need to excise it out. For this you need a sharp and distinct band of specific DNA in the gel. Therefore, to get a sharp band, the gel has to be run using the appropriate voltage.
It has been observed that 3-5V/cm is the optimum voltage for DNA extraction. Increasing or decreasing the voltage will result in poor resolution and hence the poor recovery of the DNA fragments. For extracting the DNA fragments, we generally apply a voltage of 3V/cm to the gel.
4) Cutting of the gel slice
Cutting the gel slice also has a significant effect on the recovery of the DNA molecules. You should cut the gel slice just adjacent to the band. Moreover, extra gel portions if any should be carefully removed. It has been found that extra gel portions greatly reduce the recovery of DNA molecules.
In addition, you should try to cut small gel slices for extraction purpose. Lager gel slices incompletely solubilize and hence results in the poor recovery of the DNA molecules.
5) pH of the extraction buffer
As the adsorption of DNA fragments to silica membranes largely depends on pH, therefore, extraction of DNA fragments is significantly affected by the pH of the extraction buffer. It has been observed that maximum adsorption occurs at pH less than 7.5. As the pH of the buffer increases, adsorption of DNA to silica membrane is reduced drastically.
In order to monitor the pH of the extraction buffer, many commercially available kits add indicators to the extraction buffer. If the pH increases, color of the buffer changes, thereby enabling easy monitoring of the pH.
There are certain reasons for the increase of pH of the extraction buffer. For example, agarose gel is having incorrectly prepared high pH buffer, or the electrophoresis buffer had been repeatedly used, etc. In these cases, the pH of the extraction buffer can easily be corrected by adding a small volume of 3 M sodium acetate, pH 5.2, before proceeding.
6) Residual ethanol
After the adsorption of the DNA molecules to the silica membrane, the membrane is washed with solutions having 70% ethanol. However, before the addition of elution buffer to the membrane, the residual ethanol must be removed. It has been observed that presence of residual ethanol on the silica membrane greatly reduces the recovery of the DNA fragments.
In order to remove the residual ethanol the best way is to centrifuge the column for two minutes at maximum rpm. However, we have observed that doing so doesn’t completely remove the residual ethanol. Therefore, you should keep the column at room temperature for a while to remove the residual ethanol. But be careful, as over-drying the membrane will, in turn, result in lower recovery of the DNA fragments.
7) pH and salt concentration of the elution buffer
Elution efficiency of the DNA fragments from silica membranes strongly depends on the salt concentration and pH of the elution buffer. It has been found that most efficient elution is carried out under alkaline pH (7.5-8.5) and low salt concentrations.
Generally, the DNA fragments are eluted using 10 mM Tris-Cl, pH 8.5. However, TE buffer is not recommended for the elution of the DNA because some downstream processing may get inhibited by the presence of EDTA. Apart from 10 mM Tris-Cl, pH 8.5, MQ grade water can also be used to elute the DNA fragments. However, you will have to make sure that the pH of the water remains in the range of 7.5-8.5. Moreover, recovery of DNA fragments will be less if water is used instead of the elution buffer.
8) Volume of elution buffer
Generally, elution of DNA fragments in a spin column based extraction is carried out in small volumes. However, the volume of the elution buffer plays a significant role in the recovery of the DNA molecules. It has been observed that most efficient elution is carried out using 20-50µl of the elution buffer.
Using elution buffer less than 20µl will result in poor recovery of the DNA fragments. However, using lager volumes will result in the elution of less concentrated DNA fragments. For various purposes, we generally elute the DNA fragments in 25µl of elution buffer.
9) Incubation time of the buffer on the column
Since the main purpose of the elution buffer is to provide a medium in which the DNA fragments will be dissolved. Therefore, incubating the spin column after the addition of the elution buffer has a significant role in the recovery of the DNA molecules. Generally, it is a good practice to incubate the column for 2-5 minutes prior to centrifugation.
You can clearly see that there are a number of factors influencing the extraction of DNA fragments from agarose gel using spin column based method. Each of the above listed factors should be properly considered in order to achieve efficient extraction of the DNA.
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