Since DNA is not visible to the naked eye, we stain it with either a coloured stain, such as methylene blue, or more frequently with a fluorescent stain such as Ethidium Bromide.
Fluorescent stains give much better detection levels when imaging gels. There are a wide range of stains on the market, some being added to gel before casting and some being used to stain the gel after the run. Whichever stain you use, the next step is to capture an image in a gel documentation system. Gel documentations systems , or gel docs for short, use high sensitivity cameras to capture images of the agarose gels.
Often these systems are equipped with UV or blue light transilluminators, which are used to excite the fluorescent stains, which then emit light which can be captured by the camera. All gel docs will come with some form of illumination source, a filter to remove background light and a camera to detect the signal. Other than these basics there are a huge range of gel docs available starting from basic hood systems to systems with integrated PCs and touchscreens.
Given the simple nature of this technique, scientists have been able to apply it to a wide range of studies, some of which are discussed below. Probably the most frequent application of agarose gel electrophoresis is in molecular cloning. This is the construction of recombinant DNA molecules that are integrated into various organisms to create genetic modifications. The purpose of these modifications varies and can include production of a specific biomolecule, for example the production of insulin in pharmaceutical manufacturing.
Other applications of molecular cloning include adding fluorescent protein fusions to existing cellular proteins to study their location in cells and creating new genetic circuits to carry out specific functions, such as breaking down toxins. Whatever the desired end product is, electrophoresis is a key step in both the production and quality control of DNA fragments used in molecular cloning.
Electrophoresis can be used to analyse the fragments created by polymerase chain reaction PCR or restriction digest, to ensure they are of the correct size. It can also be used to purify fragments, by running them on the gel and subsequently cutting out the band of interest and purifying the DNA from the agarose. Combined with PCR, agarose gel electrophoresis can be a powerful technique for identifying individuals based on their genetic code.
The human genome contains many regions of short repeats, the number of which vary uniquely between individuals. By targeting these regions with specific PCR primers, a profile of band on an electrophoresis gel corresponding to these regions can be created that is unique to that individual.
This technique, known as DNA fingerprinting, can be used in areas such as forensics for criminal investigations, genealogy and parentage testing. Electrophoresis can be used in a range of diagnostic tests, primarily in the screening of genetic disorders but also to identify abnormal proteins. DNA can be extracted from patients, or even from embryos for pre-implantation screening, and subject to PCR and agarose gel electrophoresis to confirm the presence of certain genes or genetic abnormalities.
Agarose gel electrophoresis can also be applied to some proteins, for example to study blood chemistry to determine suitability of certain medical treatments. The wide range of applications, both academic and clinical make agarose gel electrophoresis an extremely important technique.
Although the recent advent of next generation sequencing technologies has the potential to replace many of the current uses of agarose gels, their ease of use and versatility mean that this technique is likely to persist for the foreseeable future. The popularity of agarose gel electrophoresis is partly due to its simplicity.
The equipment required is easy to use and takes little training to operate correctly. The main components are discussed below. The gel tank, also called a gel box, is the main component of the horizontal agarose gel electrophoresis system.
Generally, a gel tank will consist of a plastic container with a raised centre platform where the gel is places on a secondary support called a gel tray. At either end of the tank, electrodes made from an inert conductive material, most commonly platinum, are fixed and wired to connectors to allow the connection to the power supply.
Finally, a lid sits on the gel tank to prevent access to the chamber while high voltage is applied to the buffer. Cleaver Scientific manufactures gel tanks in a range of sizes for different applications and can custom manufacture systems for niche applications. Take a look at the selection chart and browse our product pages for more information.
Now available with 20 x 25cm, 20 x 20cm, 20 x 15cm or 20 x 10cm gel trays Run up to samples. To apply an electrical field to the gel, you will need an electrophoresis power supply. These power supplies are specifically manufactured for electrophoresis applications and features very stable voltage and current outputs to prevent fluctuations in migrations speed.
A good power supply with allow you to set either constant current or voltage depending on the requirement of the experiment, and more advanced supplies will allow programming of individual steps at different parameter values. Agarose gel electrophoresis is one of the most important and routine techniques for DNA analysis. Combining with an organic dye ethidium bromide EB , DNA fragments could be well separated according to the nucleobase amount and expediently observed under a UV light.
The resolution of agarose gel electrophoresis for DNA separation is mainly dominated by the concentration of agarose gel and working voltage of electrophoresis. In most cases, dispersed and tailed DNA bands were obtained after electrophoresis, accompanying with serious background signals derived from EB dye. Therefore, it will be highly fascinating to develop a novel strategy to improve the electrophoresis resolution of DNA fractions with low-noise background.
Compared with the routine agarose gel electrophoresis, successive adsorption-desorption processes between DNA fragments and the surfaces of GO nanosheets dispersed in the gel net significantly improved the separation of DNA fragments with different nucleobase amounts Scheme 1. Meanwhile, the background noise derived from the diffusion of EB dye in the gel was completely eliminated because the excessive dye was adsorbed on the surface of GO nanosheets.
Then, the dispersed GO solution was added into agarose solution at designed concentrations and heated under microwave irradiation. The shift distances and width of DNA bands were measured. Numerous wrinkles were observed in the plane of the GO nanosheets Fig. The size of the GO nanosheets was calculated by measuring the area of the nanosheets and assuming it as a circle.
The inset in Fig. Four different carbon-bonding states were identified according to the peak fitting. The peaks at approximately The elimination of EB-derived background noise in agarose gel could be attributed to the adsorption of EB dye on GO sheets.
Moreover, the shift distances between different DNA bands were significantly enlarged, especially the shift between band 2 and band 3. In comparison, in the absence of GO lanes IV—VI , the broad DNA bands in agarose gel were observed, accompanied with serious background noise throughout the gel due to the diffusion of EB dye in the gel. In particular, the shift distance between band 2 and band 3 was rather small. It is generally known that the shift of DNA fragments in agarose gel was primarily depended on the nucleobase amount of DNA fragment and the voltage of electrophoresis.
The short DNA fragments shift faster than the long fragments, and the shift rate of DNA fragments is promoted by increasing the voltage of electrophoresis. The influences of GO on the shift of DNA fragments in agarose gel were investigated by adjusting the concentration of GO in agarose gel. As shown in Fig. The increased shift distance of DNA could be attributed to excellent conductivity of GO, promoting the electrophoresis rate of DNA fragments in agarose gel.
However, the further increase of the GO concentration did not continually increase the shift distances of the DNA fragment. Figure 4b shows the influence of the GO concentrations on the shift distance between the two adjacent DNA fragments. With the increase of the GO concentration in agarose gel, the shift distances between the two adjacent DNA fragments were significantly increased, implying the better separation of DNA fragments.
Figure 5 shows the influence of electrophoresis voltages on the shift distances of DNA fragments in agarose gel at the GO concentration of Figure 5b shows that the shift distances between the two adjacent DNA fragments increased with the enhancement of electrophoresis voltages, implying the better separation of DNA fragments.
Graphene and its derivate have attracted much attention for applications in DNA detection and sequencing due to its unique electronic property and single atom-layer thickness. The theoretical and experimental investigations demonstrated that single-nucleobase resolution of DNA sequencing could be realized by measuring the nucleobase-dependent transverse conductance derived from the translocation of DNA strands through the nanopores in the graphene plane [ 5 — 9 ].
Various biosensors for detection of DNA fragments and proteins have been developed based on either the extraordinarily high quenching efficiency of GO or the fluorescence resonance energy transfer between quantum dots and GO [ 12 — 18 ]. In addition, various biomolecules, including DNA, proteins, and peptides, were ready to be adsorbed on the surfaces of GO due to intramolecular interaction [ 10 , 23 ].
In the present study, DNA fragments were adsorbed onto the surfaces of GO nanosheets dispersed in agarose gel net by intramolecular interaction. The oxygen-containing functional groups in GO nanosheets could play a crucial role in the improvement of hydrogen-bonding interaction between DNA nucleobases and GO nanosheets, which is favorable to the adsorption of DNA fragments onto the surfaces of GO nanosheets.
Subsequently, DNA fragments were desorbed from the surfaces of GO nanosheets under electrophoresis condition, which could be influenced by the charges carried on both the DNA fragments and GO nanosheets. Therefore, the successive adsorption-desorption processes between DNA fragments and GO nanosheets significantly improved the separation resolution of DNA fragments by increasing the shift distances between the adjacent DNA fragments with different nucleobase amounts.
It is noticeable that reduced GO rGO nanosheets displayed size- and concentration-dependent cytotoxicity and genotoxicity in human mesenchymal stem cells, which was attributed to rGO-induced oxidative stress, cell membrane damage, DNA fragmentations, and chromosomal aberrations [ 26 , 27 ].
However, in the present studies, there is no significant increase in both the amount and width of electrophoresis bands, confirming the absence of new DNA fragments derived from GO-induced fragmentations. In addition to its usefulness in research techniques, agarose gel electrophoresis is a common forensic technique and is used in DNA fingerprinting.
Ethidium bromide is an intercalating dye, which means it inserts itself between the bases that are stacked in the center of the DNA helix.
One ethidium bromide molecule binds to one base. As each dye molecule binds to the bases the helix is unwound to accommodate the strain from the dye.
Ethidium bromide can easily get into your cells. Human DNA is linear and stains well. This means that it can get into your DNA and untwist it. This is not a good thing, so make sure you are careful and protected when using ethidium bromide.
There are also safer and less toxic alternatives that you may be able to use. The phosphate molecules that make up the backbone of DNA molecules have a high negative charge. When DNA is placed on a field with an electric current, these negatively charged DNA molecules migrate toward the positive end of the field, which in this case is an agarose gel immersed in a buffer bath.
The agarose gel is a cross-linked matrix that is somewhat like a three-dimensional mesh or screen. The DNA molecules are pulled to the positive end by the current, but they encounter resistance from this agarose mesh.
0コメント