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. The smaller molecules are able to navigate the mesh faster than the larger one, so they make it further down the gel than the larger molecules.
This is how agarose electrophoresis separates different DNA molecules according to their size. The use of dyes, fluorescent tags or radioactive labels enables the DNA on the gel to be seen after they have been separated. They will appear as bands on the gel. A DNA marker with fragments of known lengths is usually run through the gel at the same time as the samples. How is gel electrophoresis carried out?
Preparing the gel Agarose gels are typically used to visualise fragments of DNA. The concentration of agarose used to make the gel depends on the size of the DNA fragments you are working with.
The higher the agarose concentration, the denser the matrix and vice versa. Smaller fragments of DNA are separated on higher concentrations of agarose whilst larger molecules require a lower concentration of agarose. To make a gel, agarose powder is mixed with an electrophoresis buffer and heated to a high temperature until all of the agarose powder has melted.
Once the gel has cooled and solidified it will now be opaque rather than clear the comb is removed. Many people now use pre-made gels. The gel is then placed into an electrophoresis tank and electrophoresis buffer is poured into the tank until the surface of the gel is covered. The buffer conducts the electric current. The type of buffer used depends on the approximate size of the DNA fragments in the sample.
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.
At Cleaver scientific we have a range of electrophoresis power supplies for all applications. Each power supply has a 2. Constant voltage, current and power options are available as well as pre-programmed or customer programmed conditions allowing users to save and repeat their experiments for exceptional reproducibility. For the final stage of the technique, gel imaging, you will need a gel documentation system as described above.
Cleaver Scientific have a whole range of gel documentations to suite any budget or requirement. Take a look at our selection guide to find the best option for you and browse our product pages for more information. To run a gel electrophoresis experiment you will require both the equipment and the reagents. The improved resolution of DNA fragments in GO-doped agarose gel could be attributed to the successive adsorption-desorption processes between DNA fragments and GO sheets, while the elimination of the background noise could be attributed to the adsorption of the excessive EB dye on the surface of GO sheets and high fluorescence quenching efficiency of GO.
These results provide promising potential for graphene and its derivate utilized in various electrophoresis techniques for separation and detection of DAN fragments and other biomolecules. Graphene has attracted considerable attention in biomedical fields due to its exceptional electronic, thermal, and mechanical properties, as well as extremely large specific surface area [ 1 ]. It is of great interest that graphene displays promising potential in DNA analysis and detecting [ 2 — 4 ].
The theoretical calculations indicate that DNA-graphene hybrids display significant base-dependent features in the electronic local density of states derived from the different interaction energies between DNA bases and graphene, providing an alternative route to DNA sequencing [ 5 — 10 ].
Studies showed that DNA fragments were quickly adsorbed on the surface of graphene oxide GO at room temperature due to the high affinity between GO and DNA nucleobases, while the adsorption and release of the double-stranded DNA from GO were relatively slow [ 3 , 4 ]. On the other hand, graphene and its derivates were reported as the super-quenchers with the long-range nanoscale energy transfer property [ 12 — 14 ]. Therefore, GO could bind and quench a dye-labeled single-stranded DNA probe and subsequently release the fluorescent probe when it formed a duplex with its target [ 15 ].
So far, various GO-based biosensors have also been extensively developed for DNA analysis with improved sensitivity and speed [ 9 , 16 — 18 ].
For instance, a series of electrochemical biosensors with ultra-high resolution have been developed by depositing GO on the surface of graphite electrode for detection of DNA fragments at single-nucleotide base level and early diagnosis of leukemia single abnormal cell in approximately 10 9 normal cells [ 19 — 21 ]. 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.
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