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Electrophoresis is a technique that uses differences in electrical charge to separate the molecules in a mixture. Rate of migration of molecules in electrophoresis depends on its size and its charge-to-mass ratio. As DNA molecules have same shape so have similar charge-to-mass ratio, so the only separating factor of DNA molecules is their size. Simple electrophoresis cannot separate DNA fragments of different sizes efficiently; therefore Agarose gel electrophoresis is performed for DNA fragments.
What is Agarose gel Electrophoresis?
- Agarose gel electrophoresis is simple electrophoresis performed under Agarose gel.
- In Agarose gel electrophoresis, as the name suggests gel is made up of Agarose.
- Agarose is a natural polymer (complex sugar) extracted from sea weeds. It creates a complex network of pores/matrix/ like a spider web through which the DNA molecules separate.
- Agarose gel provides a sieving effect to DNA fragments of different sizes.
- As DNA molecules have negative charges, so when placed in an electric field they move towards the anode (positively charged electrode) under an electric field through a gel medium/matrix.
- The smaller the DNA molecule, the faster and farthest it can migrate through the gel.
- Agarose gel electrophoresis therefore separates DNA molecules depending upon the size of DNA fragments.
Use in Recombinant DNA technology
- DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.
- Cutting of DNA by restriction enzymes produces different DNA samples/ DNA fragments of different sizes which can be isolated by this technique and desired DNA fragment is selected and amplified by PCR (Polymerase Chain Reaction).
How to perform Agarose gel electrophoresis?
- Agarose gel is made by mixing Agarose powder with buffer solution.
- This hot melted solution is then poured into casting tray or gel setting tray, to get cool and solidify.
- Casting tray has to be fixed with combs before pouring the gel solution. Combs create wells in the gel when it gets cool.
- When cooled Agarose gel solidifies, and forms a flexible gel. Then combs are removed carefully and gel is placed in the electrophoresis chamber.
- Electrophoresis buffer is added into the chamber to cover the gel completely.
- DNA samples are mixed with bromophenol blue (blue dye) to give samples blue colour and loaded into the wells with the help of pipette.
- Electrical cords are connected to the chamber accordingly so that DNA will migrate towards positive pole (anode).
- After migration, to visualize DNA samples, DNA has to be stained with compound Ethidium bromide (which is a potent mutagen and should be handled carefully), as ethidium bromide binds to DNA and fluoresces under UV light, making DNA samples visible. One cannot see pure DNA in visible light and without staining. EtBr can be added to gel or buffer.
- Bright orange coloured bands of DNA can then be seen when exposed to UV light.
- Separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as Elution.