Molecular biology has been evolving for a long time. This field has come up with different techniques and new innovations in order to bring out the best results in the laboratory. Speaking of techniques, one of the most commonly used techniques in the lab, Electrophoresis, helps in separating charged molecules, like DNA, as per their sizes.
If you want to know more about electrophoresis and information related to it, then keep going on with this blog. You will get to know about it. Keep reading!
Gel electrophoresis is a technique commonly used for separating charged molecules like DNA, RNA, and proteins as per their sizes. These charged molecules go through a gel whenever an electric current is passed across it. There’s an electric current that is applied across this gel, so the other end has a positive charge, and the other end has its own negative charge.
This movement of the charged molecules is called migration. And the molecules are migrated towards the opposite charge. The molecule that has a negative charge will be pulled towards the positive end (opposites attract).
This gel has a permeable matrix, a bit like a sieve, through which molecules can travel whenever an electric current is passed across it. The smaller molecules are migrated through the gel more quickly. They travel further on the larger fragments, migrate more slowly, and then travel for a shorter distance. In the end, the molecules are separated by size. (“What is gel electrophoresis? – YourGenome”)
Gel Electrophoresis with DNA:
Electrophoresis enables you to differentiate DNA fragments of different lengths. Now, the DNA is negatively charged. Whenever an electric current is applied to the gel, the DNA will start to migrate towards the positively charged electrode.
Shorter strands of DNA move more quickly through the gel than longer strands, which results in the fragments being arranged in order of size.
Fluorescent tags or radioactive labels are used to enable the DNA on the gel to be seen after they have been separated. This appears as a band on the gel.
A DNA market that has fragments of known lengths is usually run through the gel, the same as the samples.
If you compare the bands of the DNA samples with those from the DNA market, then this can work out as the same length as the DNA fragments in the samples.
How The Gel Electrophoresis Is Prepared?
Preparing The Gel:
Agarose gels are used for visualizing fragments of DNA. This agarose concentration is utilized for making the gel, depending on the size of the DNA fragments you are working with.
The higher agarose concentration is directly proportional to the density of the matrix. The small quantity fragments of DNA are separated on higher concentrations of agarose.
For preparing a gel, the agarose powder is mixed with an electrophoresis buffer and heated to a high temperature until the agarose powder is melted. The melted gel is poured into a gel and then into the gel casting tray. Then, a comb is placed on one end to make wells for the sample to be pipetted into.
Once the gel has cooled and solidified, then the comb is removed. Then, the gel is placed into an electrophoresis tank, and an electrophoresis buffer is poured into the tank till the surface of the gel is covered.
Preparing The DNA For Electrophoresis
A dye is added to the sample of DNA in order to increase the viscosity of the sample, which will prevent it from floating out of the walls. (“What is gel electrophoresis? – YourGenome”)
Then, a DNA marker is loaded into the first well of the gel. The fragments in the market have a known length, so it can be used to help the size of the fragments in the samples. These prepared DNA samples are pipetted into the remaining wells of the gel.
Once this is done, the lid will be placed on the electrophoresis tank, ensuring that the gel’s orientation and positive and negative electrodes are correct.
Separating The Fragments:
The electrical current is switched on, so the negatively charged DNA starts to move through the gel toward the positive side of the gel. Shorter lengths of DNA move quicker than longer lengths. So, they move further in the time when the current is run.
Moreover, the distance the DNA has migrated in the gel can be judged visually. You can monitor the migration of the loading buffer dye. On the other hand, the electrical current is left on long enough to make sure that the DNA fragments are moved far enough across the gel to separate them.
We are pretty sure that you know about gel electrophoresis and all the information related to it. These processes are done in the lab with gel electrophoresis in order to bring accurate results.
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