VPB 321: Animal Biotechnology (2+1)

AGAROSE GEL ELECTROPHORESIS

Aim: To demonstrate the separation of DNA by agarose gel electrophoresis

Principle

• Nucleic acids are separated on agarose gel based on their molecular weights and charge.
• Nucleic acids being negatively charged, move towards anode and depending on their molecular weights, heavier fragments move slowly whereas lighter fragments fast.
• Depending upon the size of the nucleic acids to be fractioned, 0.7 to 2.0% of agarose gel is used.

Materials required

• Agarose, Boric acid, Bromophenol blue, Disodium EDTA, Ethidium bromide, Sucrose, Tris, Xylene cyanol, electrophoresis unit, UV-transilluminator

Solutions required

• TBE (Tris Borate EDTA) Buffer, pH 8.2
  • Stock Solution 10X
    • Tris Base 108 gm.
    • Boric acid 55 gm.
    • Triple distilled water to 1 lit.
    • Disodium EDTA 9.3 gm.
  • Working solution 1X
    • Mix stock solution of TBE buffer 100 ml with 900 ml of triple distilled water to prepare the working solutions of 1 X TBE buffer.
• TAE (Tris Acetic acid EDTA) buffer, pH 8.5
  • Stock solution 50X
    • Tris Base 242 gm
    • Glacial Acetic Acid 57.1 gm
    • Disodium EDTA.2 H₂O 37.2 gm
  • Working Solution 1X
    • Mix stock solution of TAE buffer 20 ml with 980 ml of triple distilled water to prepare the working solutions of 1X TAE buffer.
• Ethidium Bromide solution
  • Prepare a stock solution of 5mg / ml ethidium bromide using T?BE working solution and prepare the working solution in the concentration range of 0.5 mg / ml using TBE working solution.
• Gel loading Buffer
  • Bromophenol Blue 0.25 g
  • Xylene cyanol 0.25 g
  • Sucrose 40 g
  • 1 X TBE buffer to 100 ml.

Procedure

• Agarose gel electrophoresis is carried out in a horizontal submarine electrophoresis unit. For electrophoresis unit. For electrophoresis, 0.7% agarose gel in TBE buffer containing ethidium bromide (0.5-1 m g) is used.
• Lambda DNA digested with Hind III restriction enzyme used as molecular weight markers.
• After the addition of tracking dye, load each sample in the gel.Electrophoresis is carried out at 90V for 30 min-1 hr depending upon the length of the gel or till the bromophenol blue migrated more than half the length of the gel.
• At the end of the electrophoresis, visualize the DNA as discrete bands in the gel under UV transilluminator.

Question

• What is the purpose of using gel loading buffer in DNA agarose gel electrophoresis?
• What is the principle in ethidium bromide staining?
• What is the purpose of using UV-transilluminator in DNA agarose gel electrophoresis?