What is agarose gel electrophoresis?
- Agarose gel electrophoresis (AGE) is an approach that is used to distinguish DNA from RNA based on their molecular sizes.
- The separation of RNA and DNA molecules is accomplished when nucleic acids that are negatively charged travel through an agarose structure under an influence of an electrical fields (electrophoresis).
- In Agarose Gel Electrophoresis the Smaller molecules are more efficient and travel further than larger ones.
- This procedure is easy and quick to execute and can resolve DNA fragments that cannot be separated with other techniques like density gradient centrifugation.
- The DNA’s position in the agarose gel is revealed by staining the gel with a low amount of intercalating fluorescent dyes like Ethidium bromide.
- The procedure consists of three essential steps: Preparation the agarose gel, Electrophoresis of DNA fragments visualization of DNA fragments.
- Agarose gels can be utilized to efficiently separate fragments ranging from 50 bp to a few thousand bases long by altering the porosity the gel and the application of current.
- DNA can be purified from gels by a number of methods such as: Electroelution, electrophoresis onto DEAE Cellulose/Nitrocellulose (NA45) paper, using b-Agarase (from low melting agarose) or using glass beads/ silica etc.
Purpose of agarose gel electrophoresis
- To visualize separated DNA fragments according to their molecular size by applying an electric current to the gel matrix.
Agarose gel electrophoresis principle
- Agarose is an amorphous polymer that has been extracted from seaweeds.
- Pure agarose is that is insoluble in buffer or water at room temperature, but it dissolves upon boiling. The solution that is molten is then poured into a mould and then allowed to set. When it cools, agarose is subjected to polymerization i.e. sugar polymers cross-link each other , causing the solution to gel. The density or the size of the pore that is determined by the the concentration of the agarose.
- DNA’s charge is negative when it’s at neutral pH. When an electrical field is applied to the gel, it is moved toward the anode. The movement of DNA across the gel depends on the size of DNA molecules Agarose concentration, the conformation of DNA, applied current
- Matrix of agarose gel works as a molecular filter through the DNA fragments can move upon the application of an electric current.
- A higher concentration of agarose results in stronger gels. i.e. spaces between the cross-linked molecules are less and therefore smaller DNA fragments can easily move through these gaps.
- When the size of DNA grows, it becomes more difficult for the DNA to travel through the spaces. Likewise, the lower concentration of agarose aids in the movement more large DNA pieces since the distance between the cross-linked molecules are larger.
- The progression of electrophoresis in gels is tracked by monitoring the movement of an invisible color (tracking the dye) across the gel.
- Two of the most commonly used dyes are Xylene Cyanol and Bromophenol blue, both of which travel at the same rate as double-stranded DNA with sizes of 5000 bp and 300 bp, respectively. The dyes used to track are negatively charged low molecular weight compounds that are added to each sample prior to the beginning of the run. Once the dye is able to reach the anode, the run ends.
- Because DNA isn’t naturally colored so it won’t be evident in the gel. Therefore, the gel, following electrophoresis, gets stained by an in-specific dye for the DNA.
- Distinction bands are visible in the presence of enough DNA that binds the dye to reveal it otherwise, the band cannot be observed.
- The gel is viewed against a white background, where DNA appears as dark-coloured bands.
- Alternately you can use an intercalating dye, such as Ethidium bromide can be added to the agarose gel. The the location of bands is identified by examining the gel in UV which is when DNA reflects.
Take note: Ethidium bromide is to be handled with care since it can cause mutagenesis and is carcinogen. Use gloves when working with EtBr solutions and gels stained by EtBr.
Factors determine the rate of migration of DNA through agarose gels
The following elements influence the rate of transfer of DNA through agarose gels.
- Molecular size of DNA: Fragments of DNA that are linear move through agarose gels, with an amount of mobility that is related to their log10 molecular weight. Larger molecules travel slower because of the more frictional drag when they travel through the gel’s pores less efficiently than smaller molecules. Circular DNA forms migrate in agarose different from linear DNA with the same mass. Undigested plasmids travel faster that the identical plasmid if linearized.
- Agarose Concentration: Through the use of gels that contain different concentrations of agarose, DNA fragments that are various sizes can be separated. A higher concentration of agarose aids in the separation of small DNA and lower concentrations of agarose allow the resolution of large DNA.
- Electrophoresis buffers: Many buffers have been suggested for the electrophoresis of DNA. The most popular is The TAE (Tris-acetate-EDTA) as well as the TBE (Tris-borate-EDTA). DNA fragments move in different ways within the two buffers because of differences in the strength of ions. Buffers don’t just create the pH, they also supply Ions that help to improve conductivity.
- Effect of Ethidium bromide: it’s a is a fluorescent dye that emits light between 254-366nm. It is intercalated between the nucleic acids’ bases and permits the very efficient analysis of DNA fragments inside gels using an ultraviolet transilluminator. The binding of ethidium chloride to DNA alters its weight and rigidity and, consequently, its mobility.
- Voltage: The voltage applied to the gel increases, larger fragments move faster than smaller pieces. The most precise resolution of fragments greater than 2 kb is achieved by applying no more than 5 volts per cent on the gel (the cm value represents the distance between two electrodes, it isn’t the entire length).
Requirement for Gel electrophoresis
- Genomic DNA
- Plasmid DNA
- 50X TAE
- 1 Kb DNA Ladder
- 6X Gel Loading Buffer
- Glass wares: Conical flask, Measuring cylinder, Beaker
- Reagents: Distilled water, Ethidium bromide (10 mg/ml)
- Other requirements: Electrophoresis apparatus, UV Transilluminator, Micropipettes, Tips, Adhesive tape, Microwave/Burner/Hotplate
Preparation of 1X TAE (gel electrophoresis buffer): To prepare 500 ml of 1X TAE buffer, add 10 ml of 50X TAE Buffer to 490 ml of sterile distilled water*. Mix well before use.
Agarose gel electrophoresis protocol
Preparation of 1% Agarose Gel
- Make 1X TAE preparation by diluting a suitable amount of buffer 50X. (For an experiment of one, about 200 ml of 1XTA is needed. Create 4ml of 50X TAE up to 200 ml using distilled water).
- Measure 0.5 grams of agarose and add it to 50ml of TAE 1X. This will yield the agarose gel at 1. Cook until the agarose gel dissolves completely and clear solutions are formed.
- Then, place the combs of electrophoresis in a position that is 2cm to the anode.
- Pour the solution of agarose into the tank’s central area at the point that it reaches 60 degrees Celsius.
- Do not create air bubbles.
- Its thickness must be between 0.5 up to 0.9 cm.
- Make sure the gel is kept at room temperature to allow the agarose to set.
- Pour 1X TAE into the gel tank until the buffer level is 0.5 to 0.8 centimeters above the gel’s surface.
- Carefully lift the combs up and ensure that the wells are unbroken.
- The power cable should be connected to an electrophoretic power source in accordance with the convention red anode, and black cathode.
- The samples should be loaded into those wells, in correct order.
- Set the voltage at 50 V and then turn on the power source.
- Turn off the power once the dye that tracks (bromophenol blue) in the well is 3/4th of the gel. It takes about one hour.
Staining Procedure to Visualize DNA
- Prepare 1X staining dyes through diluting the 6X dye (1:6) with distillate water. (Approximately 50 milliliters of 1X staining dye is required for a single experiment. So, you should mix 8 ml of dye 6X up to 48 ml using distilled water).
- Make sure to transfer the gel (from the gel tank) into a tray that contains 1X staining solutions. Be sure that the gel is fully submerged.
- To achieve uniform staining, put the dish on a stand for around an hour, or shake it intermittently between 10 and 15 minutes.
- Dissolve the staining dye into the container. (The dye can be used two times). Clean the gel with tap water many times until the DNA appears as a dark spot against an unlit blue background.
Notably, Ethidium bromide could be used to visualize DNA fragments. Add Ethidium bromide to molten agarose until an amount of 0.5 Ug/ml (from an initial stock of 10 mg/ml of water) at temperatures approximately 50degC. Mix the gel and then cast it. The DNA sample are visible under UV light. They appear to be fluorescent. Destaining is not required in this situation.
Applications of Agarose Gel Electrophoresis
- Electrophoresis on Agarose gels is a method that is commonly used to separate DNA, proteins or the RNA.
- Used for the Size estimation for DNA molecules.
- Analysis of PCR products e.g. in molecular genetic diagnosis , or fingerprinting of genetics.
- Separation of genomic DNA that is restricted before Southern analysis as well as of the RNA prior Northern analysis.
- The electrophoresis of agarose gels is extensively used to determine the size of DNA fragments following digesting with restriction enzymes e.g. in the restriction mapping of DNA cloned.
- The electrophoresis of Agarose gel is typically employed to identify circular DNA that has a different supercoiling patterns, and also to determine fragments that are different due to the process of DNA synthesizing.
- Apart from being an ideal medium for analyzing the size of fragments, they also allow the purification of DNA fragments. Since the removal of DNA fragments with sizes that are separated by the agarose gel can be required for many molecular procedures like cloning and cloning, it is essential to be able to remove fragments of significance in the gel.
Observation and Result of Agarose Gel Electrophoresis
The light reflection of stained gels with UV light (254-366 nm) permits DNA banding to be seen against a background that is free of dye. The gel image is recorded by making an Polaroid(tm) image or by using the gel documentation system.
Advantages of Agarose Gel Electrophoresis
- In most cases, only one component of agarose is required and no catalysts for polymerization are needed. So, agarose gels can be easy to prepare and fast to make.
- The gel can be easily poured and does not alter the samples.
- The samples may be taken for further analysis.
Disadvantages of Agarose Gel Electrophoresis
- Gels can melt during electrophoresis.
- The buffer could become exhausted.
- Different types of genetic material can be found in a variety of types.
Revision Question and answer
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.
Agar has a lot of sulphate groups (sulfur surrounded by oxygens). These are also negatively-charged, so they can interfere with how the DNA moves through the gel. So it would make a bad matrix for electrophoresis. BUT agarose is neutral, making a good matrix for electrophoresis.
To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode.