Agarose gel electrophoresis is one of the most effective laboratory techniques to separate DNA fragments of varying sizes ranging from 100 base pairs to 25 kb. Agarose is isolated from seewees genera Gelidium and Graciloria and contains repeated agarbiose subunits.
This potential of separate molecules on the basis of size may prove highly functional in a range of research applications like identification of the unknown samples compared to known results or practicing accuracy or quality control during other techniques. In this article, we will discuss the reagents for agarose gel electrophoresis, equipment required for the process, procedure, gel electrophoresis using DNA Ladder, types of DNA ladder, formulation of chemicals used, and at the end applications of the technique.
Equipment for Agarose Gel Electrophoresis
The popularity of this process is partly due to its simplicity. The equipment needed is quite easy to handle and requires a little training to operate it correctly. The main apparatus are discussed below.
1. Gel Tank or Gel Box
The gel box, also known as a gel tank, is the primary part of the horizontal agarose gel electrophoresis experiment. It generally contains a plastic can with its little bit raised area from the center where the gel has to be place on a secondary support (known as a gel tray). The box has two ends, at either end of the box; electrodes are fixed and wired to the connectors to flow the connection to the power supply. These electrodes are usually made from an inert conductive material, most probably platinum. At the last, there is a lid that sits on the gel box to avoid access to the chamber during the time when high voltage has been applied to the buffer.
2. Power Supply
An electrophoresis power supply is required to apply an electrical field to the gel. This kind of power supply is particularly manufactured for electrophoresis purpose and characterizes very stable voltage and current outputs to avoid fluctuations in migrations speed. A power supply with good quality will allow you to set whether constant current or voltage. Most commonly, it depends on the requirement of the experiment, and also, on the more advanced supplies as they can allow more programming of the individual steps at different parameter values.
3. Gel Documentation System
For the last stage of this technique for the separation of DNA fragments, a gel documentation system is required.
Reagents for Agarose Gel Electrophoresis
In order to run a gel electrophoresis experiment, both the equipment and the reagents are required. Major reagents need to perform agarose gel electrophoresis are:
- The agarose powder, for gel formation
- Buffer stocks for running buffer formation
- Loading dye for the mixing of DNA
- DNA Ladders for the comparison of DNA lengths
- DNA stain to visualize DNA
Properties of agarose gel
Agarose gel is easy to cast and handle as compared to other gels or matrices. The nucleic acids are not chemically chanded during electrophoresis and samples are also easily recovered. After the performance is done, agarose gel can be kept in a plastic bag in a refrigerator.
This gel is a three-dimensional matrix containing helical agarose molecules in supercoiled clutters that are combined into the three-dimensional structures along with channels and pores so the other biomolecules can pass as well. The 3-D structure is stayed together by the hydrogen bonds and can therefore be deteriorated by heating it back to a liquid state. Its melting temperature is not likewise from the gelling temperature, actually it may depend on the sources, the gelling temperature of agarose gel ranges from 35 to 42 °C and a melting temperature of 85 to 95 °C.
The pore size in case of a 1% range from approximately 100 nm to 200–500 nm, so absolute strength of its gel can allow it as dilute as 0.15% to form a slab for this technique. Gels with low-concentration like 0.1–0.2%, however, are fragile and also hard to cast. Agarose gel has low resolving power than polyacrylamide gel for DNA but has a much higher range of separation. That is why it is used for DNA fragments usually of the sizes 50–20,000 base pairs. The minimum resolution for the standard agarose gel electrophoresis is about 750 kb.
How is gel electrophoresis carried out?
Gel electrophoresis is performed with the help of following steps:
- Preparation of the gel
- Preparing the DNA for electrophoresis
- Setting of gel apparatus
- Observing the separated fragments of DNA
1. Preparation of gel (0.8%)
Weight about 0.4 g of agarose into an Erlenmeyer flask. Agarose gel is made by w/v percentage solutions. The concentration of agarose in a gel will depend upon the size of DNA fragments to be separated. Then add running buffer—to the agarose containing flask. Swirl to mix it. Amount that is added to the buffer is 50 mL. TAE buffer is used as well. Melt the mixture in an oven for about 1 minute.
2. Preparing the DNA for electrophoresis
Now, add ethidium bromide (interclating dye) in 4 microliter quantity in a buffer mixture and mix it gently.
Precaution– Ethidium bromide is a suspected carcinogen and must be properly disposed. Gloves must be worn and it should be covered with aluminum foil.
After addition of ethidium bromide, allow the agarose to cool down on the benchtop and then place the gel tray into the casting apparatus. Alternatively, tap the open edges of gel tray to make a mold. Plastic comb is put on the mold to create the wells. After this, pour molten agarose into gel mold and allow the agarose to become harden at room temperature for about 30 minutes. Then, comb is removed carefully to secure wells.
3. Setting of gel apparatus
Now, the loading dye is added to samples to be separated. Loading dye helps to track how far DNA sample has travelled and also enables the sample to sink into the gel. 120 volts of the power supply is programmed now. Attach the leads of the gel to power supply. The lid is removed and DNA sample is loaded carefully.
Replace the lid to gel box. Make sure cathode should be closer to the wells than anode. Turn on power now.
4. Observing the separated fragments of DNA
When electrophoresis has completed, turn off the power and remove the lid of box. Take out the gel from gel box and withdraw the extra buffer. At this moment, expose the gel to UV light.
This step is most commonly done by using Gel Documentation System (GDS). The DNA bands will show up as orange fluorescents bands. At the last, gel and running buffer will be disposed of per institution regulations.
