Agarose Gel Electrophoresis In Agarose Gel Electrophoresis Protocol

Agarose Gel Electrophoresis
Agarose Gel Electrophoresis

Agarose Gel Electrophoresis

Agarose gel electrophoresis as we know that proteins can be separated from one another by sodium dodecyl sulfate (SDS) page on the basis. In a similar fashion, the separation of nucleic acids can be achieved in agarose gel electrophoresis and the no of nucleotides can be estimated without the use of detergent, such as sodium dodecyl sulfate (SDS) in an agarose gel electrophoresis protocol.

This is possible because a simple relationship exists between the no of nucleotides in nucleic acid and its charge at neutral pH. One negative charge exists per nucleotides residue at neutral pH so the migration of nucleic acid in agarose gel electrophoresis is dependent upon the no of nucleosides. DNA fragments of 600 or fewer base pairs can be separated in polyacrylamide gel. Larger DNA and RNA fragments can be separated in agarose gel electrophoresis protocol. These gel are mostly seen in a horizontal apparatus which was discussed in paper electrophoresis.

What Is Gel Electrophoresis?

Electrophoresis is another method of separating a mixture of an organic compound. The technique of electrophoresis is used in separating charged species like proteins (hemoglobin, serum proteins, lipoprotein, and isoenzymes). It is most useful in the diagnosis of hemoglobin pathies.

Meaning Of Electrophoresis

  • Electro – Electric Filed
  • Phoresis – Movement of migration of charged ions.

So the electrophoresis is the movement of charged ions in an electrical field. Electrophoresis is very useful as an analytical method because proteins can be separated and visualized so that different proteins can be separated.

Different Types Of Electrophoresis

  1. Paper Electrophoresis
  2. Gel Electrophoresis
(a) PAGE ( Polyacrylamide gel electrophoresis)
(b) SDS- PAGE ( Sodium do decyl sulphate – Polyacrylamide gel electrophoresis)
(c) Agrose gel electrophoresis

Medium Of Agarose Gel Electrophoresis

Agarose gel electrophoresis is polymers of polysaccharides agarose agar and agaropectin. This is a cheap, non-toxic gel. Agarose gel electrophoresis protocol are formed by a noncovalent binding between polysaccharide strands. Agar dissolved in the aqueous boiling buffer and set to form a gel at about 38 calculus gel of dimension 12x 25 cm are used for agarose gel.

Detection of Bands

Since nitric acid are colorless so it should be treated with some stains. So that they can be visualized easily. Nucleic acid can be stained by silver stain.

1. Methyl green – If give red color to RNA and blue color to DNA.

2. Ethidium-Bromide – Ethidium bromide binds to DNA. If fluoresces under UV light so the bands of DNA can be easily visualized by seeing the gel in UV light.

3. Silver Staining-Silver staining can be done for staining both DNA and RNA. It is a very sensitive method.

4. Radio-isotopic staining-A very sensitive detection method involves this incorporation of radioisotopes into DNA. Molecules p32 is often used because it can be incorporated into DNA. Phosphate and its units beta particles can be detected by autoradiography.

Application of Agarose Gel Electrophoresis

1. Separation Of DNA Molecules – A mixture of nucleic acids such as DNA and RNA Can be separated using agarose gel electrophoresis. In this technique, the DNA molecules differing only in one nucleotide can be very easily separated because the migration of nucleic acid in agarose gel electrophoresis is dependent upon the no of nucleotides usually DNA With 300-2000 nucleotides can be separated easily. This technique of pulse-field gel electrophoresis can be used to separate 16 different yeast.

2. Use In DNA Sequencing – Sequence of DNA can be determined by Sanger and Maxam Gilbert method. In both these methods of DNA sequencing agarose gel
electrophoresis is widely used for the separation of the various DNA fragments.

3. In Recombinant DNA Technology -Agarose gel electrophoresis is extremely used in recombinant DNA technology. After the cleavage of particular DNA by restriction endonuclease, Different fragments produced and these fragments can be separated from each other on an agarose gel electrophoresis

4. Estimation Of Size If Single std RNA or DNA – Agarose gel electrophoresis can be used for estimating the size of std RNA and DNA by their electrophoretic mobility is the gel. This is accomplished by running an std DNA and RNA sample and the unknown DNA& RNA. The band of unknown DNA or RNA can be compared with Std DNA and RNA and so the no of nucleotides can be estimated. (Std= sexually transmitted disease).
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