[Physics Class Notes] on Gel Electrophoresis Pdf for Exam

Gel Electrophoresis is made of two words, viz: Gel and Electrophoresis.

Here, Gel is semi-solid. They are colloids or the aggregates of fine particles dispersed in a continuous medium. A medium in which the liquid remains viscous and behaves more or less like a solid or semi-solid. 

However, Electrophoresis is the motion of charged particles in a gel/fluid under the influence of an applied electric field.

Gel Electrophoresis is a method of separating DNA fragments, macromolecules like proteins, and RNA, according to their size and charge. 

This page explains to you what gel electrophoresis is. Also, how DNA/RNA fragment separation occurs in detail.

Introduction to Gel Electrophoresis 

Assume you have quite recently done a PCR (Polymerase chain reaction), making numerous duplicates of an objective DNA region. 

On the other hand, you’ve done some DNA cloning, attempting to “glue” a quality into a round DNA plasmid. 

Presently, you need to check and see whether your PCR worked, or whether your plasmid has the right gene in it. What strategy would you be able to use to picture (straightforwardly notice) the parts of DNA?

Here, we approach it with a technique called Gel Electrophoresis.

Do you know what gel electrophoresis is? If not, let’s understand it in brief.

What is Gel Electrophoresis?

Electrophoresis involves passing a current through a gel containing the DNA molecules. 

Based on their size and charge, the molecules travel through the gel in varying directions or at different speeds. This allows them to separate from one another.

Also, all the DNA fragments carry the same amount of charge per mass. 

Therefore, gel electrophoresis of DNA fragments separates them on the basis of their size only. 

Using electrophoresis, we can ascertain how many different DNA fragments are present in a sample.

Further, we can see how large they are in comparison with one another. 

Furthermore, we can also determine the absolute size of a DNA piece by investigating it next to a standard “yardstick” made up of DNA fragments of known sizes.

How Electrophoresis Works?

In a technique of gel electrophoresis, we load a DNA or macromolecule-like RNA or proteins sample into pocket-sized indentations or wells at one end of a gel. After this, we supply an electric current to pull them through the gel.

Since DNA fragments are negatively charged, they move towards the positive electrode of the gel box. 

However, in comparison to the large fragments, the small fragments move faster through the gel.

When a gel completely stains with a DNA-binding dye, the DNA fragments are visible as bands. Here, each band represents a group of DNA fragments of the same size.

Now, let us understand how electrophoresis apparatus works in DNA fragment separation:

Gel Electrophoresis Apparatus

Firstly, we add gel to a gel box. 

Secondly, we affix one end of this box to a positive electrode and another to a negative electrode.

Thirdly, we fill the main body of the box (a place filled with a gel) with a current-conducting salt-containing buffer solution.

Additionally, we position the end of the gel with wells towards the negative electrode. 

However, we position the ends without wells toward the positive electrode. It is the end where DNA fragments will migrate.  

How Do DNA Fragments Move?

After supplying the power to the gel box, the current begins to flow through the gel. 

The fragments of DNA molecules possess a negative charge due to which the phosphate groups in their sugar-phosphate backbone start moving through the matrix of the gel towards the positive pole.

After the power is on, the current passing through the gel, keeps the gel running.

As the gel keeps on running, smaller pieces of DNA travel faster through the pores of the gel matrix than longer ones. 

The movement of gel continues and the shortest pieces of DNA reach close to the positive end of the gel.

However, the longest pieces of DNA persist near the wells. 

Even so very small pieces of DNA run right off the end of the gel if we keep the power on for a long period.

How DNA Fragments Look Like After Separation?

After the DNA fragment separation is complete, we can examine the gel and also see what sizes of bands are found on it. 

When a gel stains with a DNA-binding dye, we place it under UV light. Under the UV light, the DNA fragments shine. 

These shining DNA fragments are present at different locations along the line of the gel.

A band in the above diagram (b)  is a well-defined “line” of DNA on a gel.

Here, each band comprises several same-sized DNA fragments that have all traveled as a group to the same position. 

A single or a small group of DNA fragments are not easily visible on a gel.

By comparing the bands (from Diagram. b) in a sample to the DNA ladder ( Diagram. a), we can estimate their approximate sizes. 

For instance, the bright band of the DNA molecule on the gel is around 700700700 base pairs (bp) in size.

Examples of Gel Electrolysis         

1. TBE and Denaturing PAGE (polyacrylamide gel electrophoresis) – For RNA separation

2. TAE Agarose Gel Electrophoresis – For DNA separation           

3. SDS PAGE – Denaturing gel electrophoresis – For protein identification & separation.