U3 AOS1 Topic 10: Gel Electrophoresis

Gel electrophoresis is a technique used to separate DNA fragments on the basis of their molecular size.

Assembly of Gel Electrophoresis:

·        Electrophoretic tank with positive terminal (anode) at one end and negative terminal (cathode) at the other end allowing the electric current to pass through.

·        Buffer solution which is ion rich solution and carries electric current is added to the tank.

·        Agarose gel having tiny pores to allow the movement of DNA fragments is immersed in buffer solution.

Steps of Gel Electrophoresis:

1.     Agarose gel with wells at one end is prepared and immersed in buffer solution. DNA fragments are also prepared using restriction endonucleases and amplified using PCR.

2.     DNA fragments are added to the wells using a micropipette. A standard ladder which is a mixture of DNA fragments of known sizes is also loaded into a well. Each sample is loaded into separate well and each well has thousands of DNA fragments of same length as it is extracted from the same sample.

3.     Electric current is passed through the gel and DNA fragments being negatively charged are attracted towards the positive electrode and moves through the pores of gel towards anode.

4.     DNA fragments which are smaller in size move through the pores more easily and reach the opposite end of gel earlier than the larger fragments. After which the current is switched off and DNA fragments settle on the gel as bands.

5.     The bands are not visible and therefore are visualized under UV light after staining the gel with a fluorescent dye called as ethidium bromide.

Example:

If a person A has allele for the eye color of size 10kb long and person B has allele for eye color of size 15kb long person A’s band will be further from the well as it has moved faster whereas,  person B’s band will be near the well. If two DNA samples are of same length then bands of both samples will be at same distance from well. The size of DNA fragments can be estimated by comparing its band with the band of standard ladder.