Gel Electrophoresis

Dept. Biochemistry & Molecular

Biology FMUI
 Definition
• Gel electrophoresis is a widely used technique
for the analysis of nucleic acids and proteins.
Agarose gel electrophoresis is routinely used
for the preparation and analysis of DNA.

• Gel electrophoresis is a procedure that

separates molecules on the basis of their rate of
movement through a gel under the influence of
an electrical field.
• DNA is negatively charged.
• When placed in an electrical field, DNA will migrate toward the positive
pole (anode).
• An agarose gel is used to slow the movement of DNA and separate by size.

H O2
 

DNA

- +

Power • Polymerized agarose is porous,

allowing for the movement of DNA
Scanning Electron Micrograph of
Agarose Gel (1×1 µm) 
How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size

DNA

small
large

- +

Power
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.
 Agarose

D-galactose 3,6-anhydro
L-galactose

•Sweetened agarose gels have been

eaten in the Far East since the 17th
century.

•Agarose was first used in biology

when Robert Koch* used it as a
culture medium for Tuberculosis
bacteria in 1882
*Lina Hesse, technician and illustrator for a colleague of Koch
was the first to suggest agar for use in culturing bacteria

Agarose is a linear polymer extracted from seaweed.

Making an Agarose Gel

• An agarose gel is
prepared by
combining agarose
powder and a buffer
solution (TAE 1x)
• 1 % (1 gram in 100
mL)
 Preparing the Casting Tray

Seal the edges of the casting tray and put in the combs. Place the casting tray on
a level surface. None of the gel combs should be touching the surface of the
casting tray.
 Agarose Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.
 Melting the Agarose

Agarose is insoluble at room temperature (left).

The agarose solution is boiled until clear (right).

Gently swirl the solution periodically when heating to allow all the grains of agarose to
dissolve.
***Be careful when boiling - the agarose solution may become superheated and may boil
violently if it has been heated too long in a microwave oven.
 Pouring the gel

Allow the agarose solution to cool slightly (~60ºC) and then carefully pour
the melted agarose solution into the casting tray. Avoid air bubbles.
Each of the gel combs should be submerged in the melted agarose solution.
When cooled, the agarose polymerizes, forming a flexible gel. It should appear
lighter in color when completely cooled (30-45 minutes). Carefully remove the
combs and tape.
Place the gel in the electrophoresis chamber.
 Electrophoresis of PCR Product
1. Obtain your PCR tube,
centrifuge
2. Add 10 μl of PV92 XC
loading dye into you PCR
tube and mix gently
3. Using a clean tip for each
sample, load 20 μl of the
samples into 8 wells of
the gel
Lane Sample Load Volume
1 MMR (DNA standard) 10 μL
2 Homozygous (+/+) control 10 μL
3 Homozygous (–/–) control 10 μL
4 Heterozygous (+/–) control 10 μL
5 D1 20 μL
6 Pi.1 20 μL
7 D2 20 μL
8 Pi.2 20 μL
9 D3 20 μL
10 Pi.3 20 μL
11 D4 20 μL
12 Pi.4 20 μL
Electrophoresis of RFLP Product
 Staining Process
• Ethidium Bromide
- a DNA interchelator, inserting itself into the spaces between the
base pairs of the double helix.
- It fluorescens under UV light

CAUTION: ETHIDIUM BROMIDE IS A POTENT MUTAGEN. HANDLE

ONLY WITH GLOVES AND PROPER PRECAUTIONS.

• Fast Blast
- Fast Blue belongs to the Thiazine family of dyes
- The positively charged dye molecules are attracted to and bind to
the negatively charged phosphate groups on DNA
- DNA will stain a deep blue color with consistent results
- nontoxic
Actual Alu PCR
Results
 Analysis of Stained Gel

Determine
restriction fragment
sizes

• Create standard
curve using DNA
marker

• Measure distance
traveled by
restriction fragments

• Determine size of
DNA fragments

Identify the related

samples
 Fingerprinting Standard Curve: Semi-log

Molecular Weight
100,000

Determination

Size (bp) Distance (mm) 10,000

Size, base pairs

23,000 11.0 B

9,400 13.0
1,000
6,500 15.0

4,400 18.0

2,300 23.0
100
A
2,000 24.0 0 5 10 15 20 25 30
Distance, mm
 Paternity Test

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