Agarose Gel Electrophoresis

Aim: To perform gel electrophoresis for DNA extracted from human blood sample.

Principle
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.

Materials / Reagents

 Loading dye (4g agarose, 25mg Bromophenol blue, make up to 10ml with Distilled
water)
 Agarose 1 % (dissolve 1g agarose in 100 ml TAE buffer, heat, cool, pour solution in
electrophoresis tray until it solidifies)
 Molecular weight marker ladder
 TAE buffers (50X TAE, add 242g Tris base to 57.1 acetic acid and 100ml of 0.5M
EDTA add Distilled water to IL, adjust pH to 8.2 by KOH.
 Staining dye (0.5g / ml in agarose gel).

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 5g of red gel, mix it well and pour in
electrophoresis tray using a proper gel comb, remove the comb after gel solidified.
C- Prepare the DNA sample as follows: (don’t forget to use fresh pipette tips each time)
4 l ladder
4 l loading dye
12 l ultrapure water
D- Prepare your DNA sample as follows:
10 l of DNA
4 l of loading dye
6 l ultrapure water
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.

Results / Observations
Write lab report and record your results by photographing the gel with digital camera.

Post Lab Questions

1. What are restriction enzymes? from where they are produced? Give examples?
2. What is the chemical structure of agarose and from where is produced?
3. What is the effect of ethidium bromide on human health?
4. What are the applications of restriction enzymes in recombinant DNA technology?