Tag Archives

2 Articles

What is gel electrophoresis | Types and principles

What is gel electrophoresis | Types and principles

Gel Electrophoresis is a widely used analytical method in molecular biology and biotechnology.

Widely practiced for the separation of particles like viruses, small DNA fragments and nucleic acids and proteins. Also used after the PCR, to separate the DNA copies according to their quality.

Moreover, the electrophoresis method relies on the basic principle that the charged particles in a sample will migrate if an electric field applied to it.

Therefore, once electric field applied, the particle in the solution will migrate according to the charge or size.

Above all, widely used gels like polyacrylamide and agarose are commonly used .

This gel gives sieving or separating effect, and the particle separated by both size and charge.

Small molecules can travel faster than the larger one, also the negative charged particles will move towards anode. Similarly, positive charged particles will move towards cathode, in this way particle gets separated by size and charge.

Electrophoresis of cationic particle is called cataphoresis. Likewise, that of anionic particle called anaphoresis.

what is gel electrophoresis

Types of Electrophoresis

There are different types of electrophoresis but the most commonly and vividly used are mentioned below:

Capillary electrophoresis – molecules separated on the electrophoresis basis by using a capillary tube. To clarify, the molecules separated by running them through the capillary tubes. Accuracy higher but less quantity samples can be loaded at a time.

Native gels electrophoresis – protein run without SDS. Therefore, no proteins will get denatured in this method.

SDS – PAGE – Full form : Sodium Dodecyl Sulfate – Polyacrylamide gel electrophoresis. To clarify, SDS a detergent which can denature the protein and binds to the hydrophobic site of the molecule. Therefore, becoming the most dominant negative charge of the complex.

Here, the proteins are run through gel, polyacrylamide. Therefore, the particle density and size can be varied.

Electrofocusing gel – A gel with a pH gradient used here. As the particle travels through the gradient.

Meanwhile, it will loose protons and finally gets stucked at a point with no pH.

DNA Agarose gels – Here, agarose gels used to separate large fragments of DNA. The method used to separate according to the particle sizes. Here, no SDS needed because, DNA being negatively charged.

Above all, particle size sensitivity can be adjusted by adjusting gel density.

Sequencing gels – method used to separate smaller DNA fragments with a greater resolution. Firstly, the DNA denatured using heat and at the same time passed through polyacrylamide gel. Moreover, DNA which differ by 1 or 2 base can be separated more accurately.

The glowing DNA under the fluorescent light

Gel Electrophoresis applications

Applied for the estimation of DNA molecules.

Helps in mapping cloned DNA.

Analyzing the PCR products.

Gel electrophoresis widely used in forensics, molecular biology and genetic engineering.

Used in verification of amplified DNA using PCR.

Checking the quality of extracted DNA.

Gel electrophoresis a widely used technique, more modernized. Moreover, to determine the DNA fragment quality. In addition, used after the PCR to seperate and extract the DNA products.

So guys, do you like my work? Please let me know in the comment section. Also, do not forget to share with your near and dear ones.

Spread the science power!

What is PCR? | Polymerase Chain Reaction Explained

What is PCR? | Polymerase Chain Reaction Explained

What is PCR?

Polymerase chain reaction is a technique through which the DNA is ampified and desired copies of the DNA is extracted.

What’s with the name?

Polymerase stands for enzyme, chain means the type of reaction. PCR is a chain reaction. Therefore, it is an Enzyme linked chain reaction, which makes copies of the DNA.

PCR – Uses

This technique is used to diagnose infections, above all to detect viral infections.

For instance, The blood sample of the patient is taken and the viral DNA is extracted and amplified.

Used in crime investigation, as it involves DNA. Also, in branches of Biotechnology, Forensic and molecular biology.

Used in Genetic research

Materials required in PCR

Now, you have understood what is pcr and it’s uses. To run a reaction ingredients are required, so here are the ingredients or materials required for the PCR:

1) Thermal cycler or PCR machine

To understand the PCR, first of all it is necessary to understand what is PCR machine. PCR machine or thermal cycler machine is where the reaction takes place. Above all, it is the main machinery in which the DNA copies are made.

what is pcr

Contains small tubes to which the ingredients mentioned below are added.

2) Taq polymerase

The DNA polymerase used here in PCR, it is extracted from a bacteria called Thermus aquaticus which can withstand high temperature. Similarly, this enzyme too can withstand high temperatures.

It is ideal for PCR, because Taq polymerase is active around 70 degree celsius. Also, it is very heat stable.

what is pcr

3) Primers

Primers are short sequence nucleotides. It provides a starting point to initiate the reactions. Moreover, it helps to choose an exact well defined portion to amplify.

3) Nucleotides

These are the basic units of DNA. Required here to make copies with the denatured parts of the DNA.

4) DNA template

This is the original segment of DNA we want to amplify, the target DNA. To clarify, the amplification starts in this template.

Now, we are set with the ingredients. In addition, we are left to initiate the reaction.

what is pcr

The process of PCR is split into three parts:

Denaturation, Annealing and Extension

Here, is the explanation step by step:

Denaturation

The first step in PCR in which the temperature raised to 96 degree celsius. In other words, done to seperate the strands of the original DNA template. That is, one DNA template into two. Here, we will get two separate strands of the DNA.

Annealing

In this second step, the machine temperature is reduced to 55 degree celsius.

After that, it results in the primer attachment to the target sequence of the stranded DNA or denatured DNA,

Extension

The last step in which the free nucleotides are required.

The temperature is increased to 72 degree celsius, meanwhile , it results in the activation of Taq polymerase. It will extend the primers to make new DNA strands.

These are the steps and the reaction occurs like a chain in which from one DNA we get two copies, further from two we get Four copies and so on, so for 100 reactions it can produce billions of copies.