Agarose Gel Electrophoresis

First semester 2020/2021

Dr. Muna Taha
Aim: To perform gel electrophoresis for DNA
extracted from human blood sample.
Agarose gel electrophoresis:
Is the most commonly used method in separating DNA molecules
according to size.
The term electrophoresis describes the migration of charged
particles in the electric field, these particles will migrate either to
cathode or to anode according to net charge nature. Agarose is a
linear polysaccharide made of agarobiose repeated units made of
galactose and 3,6- anhydrogalactose, agarose isolated from
marine algae.
Restriction (endonucleases) enzymes, also called as molecular scissors,
are enzymes cut DNA at a specific target, bacteria use these enzymes to
restrict (protect) their DNA from invading bacteriophages (viruses invade
bacteria and damage them), each enzyme has its own unique name
derived from the genus, species, and strain of bacteria produce them,
followed by a number that refers to chronological discovery order.
Applications
Restriction enzymes are powerful tools used in many, biochemical,
genetic, and recombinant DNA protocols:
1. Estimation the size of DNA molecule following restriction digestion.
2. PCR product analysis in fingerprinting.
3. Separation of restricted genomic DNA or RNA.
Factors affecting migration and restriction enzyme activity

• Temperature: most restriction digestion take place at 37C0.

• Length of the DNA molecule.

• Buffering system: optimum buffering system between pH 7-8.

• Increasing agarose concentration.

Buffers
Most buffers used are:
• TAE (Tris Acetate EDTA) buffer.
• TBE (Tris Borate EDTA) buffer, prepare 5X buffer by adding
0.45M Tris Borate to 10mM EDTA, or by adding 10.8g Tris base to
5.5g Boric acid and 0.744g EDTA in 200 ml DW, adjust pH at 8.2.
• Sodium borate buffer.
Protocol

A. Prepare 300 ml of 1X TAE buffer from 50X TAE buffer.

B. Prepare 1 % agarose gel by weighing 0.5 g agarose and dissolving

it in 50 ml of TAE buffer, heat it for dissolving, cool it to 60 °C, add
5µg of red gel, mix it well and pour in electrophoresis tray using a
proper gel comb, remove the comb after gel solidified.
Protocol
C. Prepare the DNA ladder sample as follows: (don’t
forget to use fresh pipette tips each time)
4 µl ladder
4 µl loading dye
12 µl ultrapure water

D. Prepare your DNA sample as follows:

10 µl of DNA
4 µl of loading dye
6 µl ultrapure water
Protocol
E. Load the DNA by micropipette into the gel wells.

F. Close the gel electrophoresis box and connect the apparatus to

power supply, set power between 75-150 volts, run electrophoresis for
60-90 minutes, then switch power supply off.

G. Visualize the gel under UV light.