Monday, 21 December 2020

Gel-Electrophoresis principle and Application

  Gel electrophoresis is a method of separating large molecules like segments of DNA. When we introduced some concepts in biotechnology, for make recombinant DNA plasmids and insert them in bacteria to produce many copies of a gene, or many copies of the protein produced when a gene is expressed.

We want to cut up the plasmid and analyze it, to make sure it is being copied as desired. Or imagine any other scenario where we have a mixture of DNA fragments that we want to separate and visualise. Gel electrophoresis enables us to do this.
 

Principle of Electrophoresis :

In electrophoresis there is a tray. In this tray there sits a slab of either polyacrylamide or agarose gel, which is immersed in an aqueous buffer solution. At one end of the gel there are a series of wells, and a number of samples can be loaded into these wells, each of which is a mixture of some DNA molecules of varying length.


This apparatus is also equipped with electrodes at each end, with the cathode, or negatively charged electrode, at the end where the wells sit, and the anode, or positively charged electrode, at the other end. Once everything is loaded and ready to go, the current is turned on.

Phosphate groups line the DNA backbone, and that each phosphate group contains one oxyanion, and thus carries a formal negative charge. We can therefore say that DNA molecules are negatively charged.

This means that the negatively charged cathode will repel the DNA molecules, and they will begin to travel along the gel, towards the positively charged anode, to which they are attracted.

The thing is, this gel is a sticky, porous substance, and the molecules have to migrate through the pores to move along the gel, in a process called sieving.

The larger the DNA molecule, the more difficulty it will have in navigating through the pores, which means that smaller DNA molecules will travel greater distances greater distances through the gel, while larger ones will travel shorter distances through the gel, in the same time interval.

This process is so reliable and quantifiable, that we can plot the approximate number of base pairs in a DNA molecule as a function of the precise distance it travels during gel electrophoresis.

  Once separation is complete, the current is turned off, and a DNA-binding dye is added to the system that glows a fluorescent pink in UV light. This is how the data is gathered, which will show up as thin bands that sometimes resemble a ladder, if many different DNA molecules were present in the sample, and each band contains thousands of identical DNA molecules of that particular length.

Remember, the farther away from the well a band shows up, the shorter the DNA molecule is that has produced that band.


PREPARATION OF 1.5% AGAROSE GEL AND ELECTROPHORESIS :

(Note : For a standard agarose gel electrophoresis, a 0.8% gel gives good separation or resolution of large 5–10kb DNA fragments, while 2% gel gives good resolution for small 0.2–1kb fragments. 1% gels is often used for a standard electrophoresis.)

1. Take 300mg of agarose and dissolve it in 20 ml of 0.5X TEB buffer to prepare 1.5% Agarose gel. 

2. Boil until the Agarose is completely dissolved and no obvious particles of the Agarose remain in the suspension. 

3. When the gel temperature is around 40° C, add 2ul of ethidium bromide(EtBr) and mix properly. 4. Seal the gel-costing/running tray on two sides; place the comb in the gel-tray in appropriate place, 

5. Pour the Agarose solution into the gel-costing/running tray-containing comb. 

6. Allow the Agarose to solidify in the tray, then remove the seal from the two sides of the tray without disturbing the gel. 

7. Then keep the gel-tray in the tank containing 0.5X TEB buffer with the wells in the cathode (negative side). The buffer level in the tank should be maintained above the gel tray. 

8. Gently lift the comb without damaging the wells; the gel is now ready for loading. 

9. Connect the cords between electrophoresis tank and the power pack before loading the samples. 

10. To prepare samples for electrophoresis, add 5μl of gel loading dye in to the restricted sample and mix well by pipetting. Load 15-20μl of the sample in the respective wells. 

11. In the nearby well load 3μl of DNA marker provided. 

12. After loading, switch on the power pack and adjust the voltage to 50V – 100V. 

13. Continue the electrophoresis until the dye reaches to 1/3rd of the gel or above. 

14. Observe the DNA bands by using UV Transilluminator.


Applications of Gel Electrophoresis

• This technique may be used to chop up plasmids with restriction enzymes and analyse the results. • It can be used to assess the products of gene amplification using the polymerase chain reaction.
• It can be used to isolate a specific DNA molecule of a mixture for sequencing or further characterization via a technique called Southern blotting.
• In addition to separating mixtures of DNA according to length, this technique can also be used to separate mixtures of proteins according to electrical charge, which offers information about the identity of the side chains, or a number of other very useful applications.
• The simplicity and immense utility of gel electrophoresis make it a very important part of any molecular biology laboratory today. 

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