AGAROSE GEL ELECTROPHORESIS

Hello everyone,

Today I will be writing few important points about Agarose Gel Electrophoresis,  as this the basis technique employed in Microbiological laboratory .  All the student of Microbiology should know this technique , So are You all ready to learn about Agarose Gel Electrophoresis 

The term electrophoresis  describes a   migration of charged ions under  electric field. The charged particles of various biological molecules will migrate either  to the  cathode or to the anode depending on the nature  of  their net charge. The equipment  required  for electrophoresis  consist basically of  two items  a power pack and electrophoresis unit

A power pack supplies a direct current between the electrodes in the electrophoresis unit. Electrophoresis units are available for running  either vertical or horizontal gel systems.

Agarose is a linear polyacrylamide (average relative molecular mass about 12000)made up of the basic repeat unit agarobiose,which comprises alternating units of galactose and 3,6-anhydrogalactose

Agarose gels are made by suspending dry agarose in aqueous buffer (TAE),then boiling the mixture until a clear solution forms. This is poured and allow to cool at room temperature to form a rigid gel.The gelling properties are attributed to both inter -and intramolecular hydrogen bonding  within and between the long agarose chains. This cross-linked structure gives the gel good anticonvectional properties. The pour size in the gel is controlled by the initial concentration of agarose,the large pour sizes are formed from low concentrations and smaller pour size formed from higher concentrations.

Agarose is used in gel electrophoresis to separate nucleic acids (like DNA) by size, charge and other physical properties. Gel electrophoresis uses an electrical current to make particles move. For example, DNA is negative, so it will travel towards to positive electrode of the gel box.

Agarose has small pores through which a DNA can travel. Bigger fragments of DNA travel shorter distances, because it takes longer for them to navigate through the pores of the agarose gel. Identically sized pieces of DNA will travel the same distance, which is why we get bands (DNA with loading dye) after we run a gel

PROCEDURE

1)Pour the gel slowly into the tank,leave to set for at least 30 minutes, preferably 1 hour, with the lid on then add Ethidium Bromide.

2)Pour TAE buffer into the gel tank to submerge the gel to 2–5 mm depth. This is the running buffer

3)Add an appropriate amount of loading buffer with DNA sample which will be used as sample in this electrophoresis

4)Load the first well with marker,then other wells with different samples of DNA

5)Close the gel tank, switch on the power-source and run the gel at 5 V

6)Monitor the progress of the gel by reference to the marker dye

Advantage

Relatively inexpensive, non-toxic, does not cause the sample to denature, so at the end of the electrophoresis procedure, precious or limited samples can still be recovered and used for other experimental procedures. It can give very good resolution and separation of large from small molecules as the agarose gel's pore size can be specified by the user, and only requires small amounts of sample. The process is fast (minimum of 30 minutes), and the apparatus is easy to set up and operate.

Disadvantage

If there is an increase in temperature there is chances in genetic material can adopt the shapes which are not needed.If the molecules are very similar in size, then it may be difficult or impossible to resolve or separate them from each other. Quantification of the amount of a molecule by observing the size or intensity of fluorescence of a band may be difficult.

Applications

• Separation of RNA and protein molecules
• Forensic science
• DNA fingerprinting
• Recombinant DNA technology
• Cloning
• Used in southern and northern blotting