Gel Electrophoresis

Introduction

• Gel electrophoresis is a technique used to separate DNA fragments based on size.
• Involves the movement of charged molecules in an electric field through a gel medium.

Definition and Principle

• Gel Electrophoresis: A technique that separates DNA molecules based on their sizes.
• Charge of DNA: DNA is negatively charged due to phosphate groups in its backbone.
• Gel Used: Typically, agarose gel is used, derived from seaweeds.

Process of Separation

• Separation occurs based on the size of DNA fragments, analogized to a sieve.
  • Smaller fragments pass through gel pores faster than larger fragments.
• Gel structure is mesh-like, with constant pore size.

Setup Requirements

• Complete setup includes:
  • Casting tray and gel
  • Comb for making wells in the gel
  • Electric supply
  • DNA sample with different-sized fragments
• Wells are formed near the negative terminal (cathode) to allow DNA to move towards the positive terminal (anode).

Loading and Running the Gel

• Buffer: Added to the apparatus for better conductivity.
• Loading Dye: Added to the DNA sample to track movement; it travels faster than the DNA.
• Power supply is turned on after loading, and DNA moves through the gel.

Observation of DNA Bands

• Ethidium Bromide: Used to bind to DNA and visualize it under UV light, producing bright orange bands.
  • Larger fragments lag behind, smaller fragments move faster.
• DNA Ladders: A standard chart used to compare and determine the size of DNA fragments based on band position.

Extraction of DNA

• Desired DNA fragments can be cut out from the agarose gel and extracted, a process known as elution.
• Extracted DNA can be used for further downstream processing.

Conclusion

• Gel electrophoresis is a crucial technique in recombinant DNA technology.