Have you ever wondered how a targeted DNA fragment is obtained from a complex double helix DNA strand? What is the need to obtain a specific DNA fragment? Yes, there is a technique known as “GEL ELECTROPHORESIS” to obtain the desired DNA, RNA and protein fragment. Specific fragmentation is required for the biotechnological research and studies. Now let’s get a look into what actually Gel Electrophoresis is.
|Double stranded DNA|
What is Gel Electrophoresis?
The definition says that “it is a technique that is utilized to separate DNA, RNA or protein fragments according to the size”. The word in the definition “according to size” is very important. This technique basically separates the fragment based on their size. There are various other attributes associated with the technique which shall be discussed later.
Overwiew of gel electrophoresis
Gel Electrophoresis Steps:-
Preparation of Agarose gel
Preparation of casting tray. (The sides of the casting tray are to be sealed with sellotape)
Pouring the gel.
Place the gel in the electrophoresis chamber.
Add a buffer to cover the wells.
Preparation of sample.
Load the sample in the gel.
Run the gel.
How does Gel Electrophoresis work?
To understand the working mechanism first of all we need to list down some major components of the system: Gel, Electric current supply, casting tray, combs, ethidium dibromide, buffer, bromophenol blue, polyacrylamide (for proteins), test molecule- DNA,RNA or Protein.
Initially, the gel is selected based on the test molecule. The gel is casted in the casting trays and wells are prepared with the help of combs to load the test molecule. The test molecule is loaded and the electric current is supplied. The test molecule will have a charge and so it will move towards the opposite charge. Separation will be done based on the size of the fragments.
Gel Electrophoresis of DNA:-
For the DNA gel electrophoresis agarose gel is used. Agarose gel is prepared from agar that is obtained from algae. The gel is utilized to slow down the movement of test molecules. Also the gel should be slightly porous to allow the controlled movement.
The gel is stained with some dye. Mostly, ethidium di-bromide is used for staining the gel. There is a need to stain the gel to get the visualization within the UV light source.
Note: Ethidium di-bromide is a toxic mutagen. Precautions should be taken care while working with it.
|After running the gel it is placed in UV transilluminator for visualization|
The sample of DNA is homogenized and inserted and loaded with a loading buffer i.e bromophenol blue and glycerol. Bromophenol blue is to give color to the sample and glycerol is used to increase the weight so that the sample gets inside the well.
The sample with bromophenol blue and glycerol is added into the wells through a micropipette. This step is to be performed with extra care because the gel might get disturbed. Also, care should be taken so that the sample is expelled into the well only.
After insertion the gel and wells are covered with enough buffer solutions. There are two main purposes for the pouring of buffers- (1) to maintain the pH and (2) to constantly generate ions for the smooth functioning of the Gel Electrophoresis. There are two important buffers which are utilized- Tris Acetate EDTA (TAE) and Tris Borate EDTA (TBE). TAE is utilized for high molecular weight and TBE is used for low molecular weight.
After, the test molecule is inserted into the wells electric current is applied to the gel. The system is to be properly covered with the lid. As the DNA is negatively charged, it will start moving towards the positive electrode. Gel Electrophoresis can be used to separate the molecules by size, shape and charge.
Factors determining the rate of movement across the gel:-
Size of the test molecule- big size of the molecule will not allow it to move faster. Thus, we can say that smaller the size, faster the movement.
Charge on the molecule- charge on the test molecule will determine the rate of movement. According to the formula F=q1q2/4πε.r^2 more the charge on the molecule, faster will be the movement.
Shape of the molecule- shape is also a determining factor for the rate of movement of the molecule. Considering the accurate shape of the molecule will be a complicated process and so for understanding purposes if there are two options- spherical and linear, obviously the spherical one will move faster as it has concentrated mass.
Applied electric field- optimum electric field conditions are needed for the accurate results. If the applied electric field is too high, the test molecule will move faster and will come out of the gel and vice versa. Thus, we need to optimize the electric field according to our need.
Density of the Agarose gel- if the gel is dense, it will not allow the movement of molecules. Thus, optimum density of the gel is required for the better separation.
Safer alternatives for Ethidium di-bromide:-
There are various safer alternatives for ethidium dibromide. Some of them are as described below:-
BIORad-Bio safe DNA stain.
Carolina blue stain and many more.
As each coin has two faces, the same is the case with these alternatives. These alternatives have some advantages and certain disadvantages too.
These are less expensive.
Less toxic than Ethidium dibromide.
Creates less hazardous waste.
No UV radiations are required for detection.
These require longer staining and destaining time.
Requires more DNA for the experiment to be performed.
These dyes are less sensitive than Ethidium dibromide.
Gel Electrophoresis SDS PAGE:-
Gel Electrophoresis SDS PAGE is the technique utilized for the Gel Electrophoresis of Proteins. First of all, let us understand the terms. SDS means Sodium Dodecyl Sulfate and PAGE means Polyacrylamide Gel Electrophoresis.
We all know that proteins are polymers of peptide chains. Such proteins become functional only after they get folded. In Gel electrophoresis technique, we require a single strand of protein for the analysis. So, to get the single strand of protein we need to add SDS in the protein molecule. SDS is a detergent which helps in breaking the folded structure of protein.
For the Gel Electrophoresis of protein, Polyacrylamide is used as gel instead of agarose gel because Polyacrylamide gels are used to separate shorter nucleic acids, generally in the range of 1−1000 base pairs, based on the concentration used.
Also one other thing which is different from Gel Electrophoresis of DNA is that the casting tray used for proteins is vertical whereas in the case of RNA and DNA it is horizontal. To know the reason you can click here, they have explained very well.
That’s it for Gel Electrophoresis. I hope you guys enjoy learning about it. We are here to make the understanding more clear and with an easy language. If you want us to write on a specific topic do send us your query through our contact page. Thank you so much guys.
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