Why agarose gel is used

Agarose Gel Electrophoresis. Scientists, teachers, writers, illustrators, and translators are all important to the program. If you are interested in helping with the website we have a Volunteers page to get the process started. Digging Deeper. Digging Deeper: Depression and the Past. Digging Deeper: Germs and Disease. Digging Deeper: Milk and Immunity. Couldn't you just dye? Moving through matrix The phosphate molecules that make up the backbone of DNA molecules have a high negative charge.

View Citation You may need to edit author's name to meet the style formats, which are in most cases "Last name, First name. Modern Language Association, 7th Ed. How much water are we using per year in the Phoenix area? Share this page: Share to Google Classroom. The stained agarose gel can be imaged and used for downstream analysis. For agarose gel electrophoresis of DNA and RNA samples, agarose gels are stained with an intercalating dye, which inserts itself in between the bases of the nucleic acids.

Ethidium bromide or SYBR Green are examples of fluorescent intercalating dyes that emit fluorescence when they are intercalated in the backbone of the molecule and exposed to short-wave ultraviolet UV light.

The UV-exposed stained gel can be imaged for record or downstream analyses. Agarose gel electrophoresis has been applied to accommodate several types of analyses, for example:. The isolation of nucleic acids constitutes one of the most important laboratory works. Oftentimes, the quality and quantity of the isolated nucleic acids are determinant to the success of the downstream processes. In the case of RNA, the integrity of the isolated RNA can be determined using denaturing or nondenaturing, high percentage agarose gel.

After electrophoresis, the presence of three distinct bands on the gel represents ribosomal RNAs, which can be used as an indication that the extracted RNA is intact. Agarose gel electrophoresis is suitable for the separation of small, medium and medium-large DNA fragments obtained from restriction digestion and polymerase chain reactions.

Based on the range of the expected size of the separated fragments, the agarose concentration can be adjusted Table 1. The separated DNA fragments can be recovered from the agarose gel after electrophoresis for subsequent works such as molecular cloning, making the technique especially practical. Table 1 The concentration of agarose gel and the suitable range of linear DNA fragments.

The properties of agarose which allows it to accommodate samples of various ranges of molecular sizes have made agarose gel electrophoresis one of the essential techniques in biotechnology. As an Amazon Associate Conductscience Inc earns revenue from qualifying purchases The modern pipette has had a colorful history as a standard tool in the. Stereotaxic Accesories. Conduct Lifestyle Grants Academia.

Quote Laboratory Techniques , Protocols , Science. Agarose Gel Electrophoresis. Agarose as a matrix for gel electrophoresis Agarose is a type of natural polysaccharide isolated from red seaweed that is used to make a gel. Agarose polymers form sieving pores when gelled Agarose is isolated from the agar of various red algae species, known as Rhodophyta. Preparation and setup of agarose gel electrophoresis Agarose gel electrophoresis can be broadly divided into the following steps: 1.

Agarose gel preparation: Agarose powder can be dissolved in an electrophoresis running buffer and heated. Sample preparation and loading: Samples used in agarose gel electrophoresis are in a liquid state. Electrophoretic separation: An agarose gel electrophoresis unit is composed of an agarose gel block or slab submerged in the electrophoresis running buffer placed in a closed electrophoresis chamber.

Visualization and downstream analysis: Electrophoretic separation in an agarose gel results in a migration pattern of the sample ladder and of the components in the samples. Applications of agarose gel electrophoresis Agarose gel electrophoresis has been applied to accommodate several types of analyses, for example: 1.

Quality control and quantification of nucleic acids The isolation of nucleic acids constitutes one of the most important laboratory works. Separation of DNA fragments Agarose gel electrophoresis is suitable for the separation of small, medium and medium-large DNA fragments obtained from restriction digestion and polymerase chain reactions.

In conclusion All in all, the strength of agarose gels and the simplicity in setting up electrophoresis contribute to the popularity of the technique in research and routine analysis. References Jorgenson, J. Analytical Chemistry, 58 7 , AA. Wiley-VCH Verlag. Barril, P. Magdeldin Ed. Rijeka, Croatia: InTech. Result of the electrophoresis by using INA S-7 agar. Generally, agarose of low purity is more breakable because of its low gel strength.

This disadvantage was critical especially when Southern or northern blotting was achieved in the experiment. In recent days, such blotting techniques have given way to the other; for example, polymerase chain reaction PCR to see DNA polymorphism and real-time PCR to see gene expressions. The major visualizing way of DNA in agarose gel is to use ethidium bromide EtBr or the other DNA intercalators that make fluorescence excited in certain wavelength [ 12 ].

Several protocols for staining reagent to intercalate DNA are known; a add the reagent in the gel before solidifying, b add the reagent in the loading buffer at electrophoresis, and c soak the gel in the reagent buffer after electrophoresis Figure 4. In a and b , a photograph of the gel can be taken with a gel tray, when the tray is clear.

In such a situation, a low gel strength does not disturb electrophoresis and DNA visualization. Based on these reasons, a low gel strength seems not a too much annoying point when simply doing electrophoresis and taking photographs. Scheme for staining and visualizing of DNA in agarose gel electrophoresis.

It is known that used agarose gel is reusable again and again. Recycling of agarose after electrophoresis is very effective for cost-saving. Several reports are published [ 13 , 14 ], in which used agarose gels are simply boiled and poured to a gel tray.

After cooling to make the recycle gel solid, the recycled gel is enough for applying another electrophoresis. EtBr is well known as toxic mutagen [ 15 , 16 ], so when used and stained gel is boiled, toxic fumes containing EtBr appear. This fume will be hazardous when incorporated through the respiratory system. To avoid such hazardous fumes, a freeze-and-thaw of used agarose gel is very effective for removing toxic EtBr from the gel [ 17 ].

By repeating freeze-and-thaw, the EtBr concentration of used agarose gel dramatically reduces to as much as a negligible level. The result of electrophoresis by recycled agarose is shown in Figure 5. Agarose is hydrolyzed in acidic condition. Therefore, repeating the boiling and melting step in acidic condition might degrade the polymer structure of the agarose.

The freeze-and-thaw method mentioned above is free from such a degradation. It is said that TBE has an advantage to fractionate small length DNA; in an old sequence analysis, a combination of acrylamide gel and TBE buffer was a standard condition. This is because of the difference of the price of acetic acid and boric acid. Anyhow, daily agarose gel electrophoresis is achieved in a condition of using TAE in standard. Yet another electrophoresis buffer is SB buffer, which is obtained from sodium borate.

In my experience, DNA is well migrated and fractionated in the agarose gel electrophoresis with SB buffer, although small but many air cavities appeared after finishing the electrophoresis. SB buffer resulted in small but many air cavities in the gel. It is a very simple and effective idea of cost-saving that dilution of the buffer is available or not.

In my experience, 0. Agarose gel electrophoresis with 0. A low concentration of ions in the buffer results in higher resistance in an electric circuit, which leads to heating of the buffer. Therefore, too much dilution of the buffer might result in boiling of buffer and melting of agarose gel. One of the major reasons of this higher cost is that platinum is used as electrodes in the tank. Platinum is a precious and noble metal, which is very stable and never degraded in electrolysis.

The second reason is that the buffer tank of the equipment has a special shape. Generally, the bottom face of the buffer tank has an anti-U-shaped structure Figure 8.

The third reason is that the equipment is sold with the special power supply. A horizontal view of typical buffer tank for agarose gel electrophoresis. The anti-U-shaped structure is not always necessary in buffer tank. Basically, a structure of an electrophoresis tank can be much more simple, and we can make it by do-it-yourself DIY Figure 9. DIY buffer tank for agarose gel electrophoresis. Note that the basket has a flat bottom, unlike a standard commercial buffer tank. A plastic basket in variety store so-called yen shops, 99 cents store, Dollar store, etc.

Plastic tape is put on the basal plane in three- to fourfold repeatedly, which works as a stopper of the gel during electrophoresis Figure 9. Although carbon stick like a lead of a pencil works as an electrode, stainless steel wire in hardware stores is a good choice of electrodes for agarose gel electrophoresis.

No expensive metal is needed; almost the cheapest one will be worth testing. Wireframe of mm in diameter leads to a good result.