Question

What separation method commonly uses a horizontal gel electrophoresis apparatus? Why?

Answer 1

There are two major approaches mainly used for the separation of the biomolecules, one is vertical gel electrophoresis method (SDS-PAGE for proteins and native gel electrophoresis for DNA also) and another one is horizontal gel electrophoresis.

In horizontal gel electrophoresis, DNA molecules are separated on the basis of molecular weight of DNA samples on agarose gel. In horizontal gel electrophoresis, different concentration of agarose gel can be prepared on the basis of the DNA size like if DNA is small then higher agarose concentration will be required (2% or more) and if size of DNA sample is high then 1% agarose is enough to separate DNA on the gel through electrophoresis (minimum 0.75% gel). Agarose forms a matrix where DNA molecule can move during electrophoresis from anterior to posterior side wherein higher molecule DNA move slower as compared to low molecular weight DNA. Separate DNA can be visualized after staining gel with ethidium bromide (inter-chelating agent), which can illuminate light under ultraviolet light (UV light). Horizontal gel electrophoresis is also being used for RNA samples also to check purity of RNA.

Apparatus required:

1.     Gel casting tray

2.     Microwave or hot plate

3.     Electrophoresis unit

4.     Power pack supply

5.     Gel documentation or UV trans illuminator

Reagent

1.     Agarose, routine use

2.     DNA or RNA samples,

3.     DNA marker (ladder)

4.     DNA loading or tracking dye

5.     Ethidium bromide

6.     Electrophoresis buffer (1x TAE or TBE buffer)