Gel Electrophoresis

Mr. Davis
 Electrophoresis Terms
Agarose Gel – Basically a linear polymer
extracted from seaweed that contains a pore
size of (2000 Angstroms).
• Used to separate DNA fragments according
to size
• Agarose gel matrixes help “Catch” DNA
molecules as they are transported by the
electric current.
TBE Buffer – (Tris/Borate-EDTA) – a buffer
solution containing a mixture of Tris base,
Boric Acid, & EDTA.
• TBE Buffer is used in procedures involving
nucleic acids.
Electrophoresis Process

• “Electrophoresis” the process

that refers to electrical
charges carried by the DNA
molecules. In the case of
DNA, the phosphate groups
on the sugar-phosphate
backbone of the DNA
molecule readily give up their
[H+] ions leaving the DNA
fragments negatively charged
in the TBE (Tris-Borate
Buffer) Solution.
Electrophoresis Process

• Under the influence of an

electric field, DNA
molecules migrate away
from the negative
electrode, known as the
Cathode and move
toward the positive
electrode of the Anode.
Gel Electrophoresis
Run
 Plant DNA Gel Electrophoresis Results

At the beginning of the electrophoresis run, DNA of

all length are relatively close together, As time
passes, the rate of DNA fragment migration depends
on:
• Longer DNA fragments take more time to move
through the pores of the agarose gel, so they move
slowly.
• Shorter DNA fragments migrate faster
• Size of the DNA molecule
 Applications of Gel Electrophoresis
1. DNA Typing – only method available for separating DNA fragments
suitable for Forensic DNA Typing.

2. Drugs – Capillary Electrophoresis is the most sensitive method for drug

detection.

3. Explosive Residues – Capillary Electrophoresis is more sensitive and

has a higher resolution than HPLC.
Applications of Gel Electrophoresis
4. “HPLC” –High Performance Liquid Chromatography – is a
form of column chromatography that pumps a sample mixture or
analyte in a solvent (mobile phase) at thigh pressure through a
column with chromatographic packing material (stationary phase).
The sample is carried by a moving carrier gas stream of Helium (He)
or Nitrogen (N) gas.
• HPLC – has the ability to separate and identify compounds that are
present in dissolved in liquid in trace concentrations as low as ppt
(parts per trillion).
Applications of Gel Electrophoresis
5. Capillary Electrophoresis analytical technique separates ions
based on their electrophoresis mobility with the use of an app;ied
voltage.
• The electrophoretic mobility depends upon:
a. Charge of the molecule
b. The viscosity of the gel
c. The atomic radius – the rate at which the particle moves is directly
proportional to the applied electric field, the greater the electric
field strength the faster the mobility.
Applications of Gel Electrophoresis
d. Agarose concentration
e. DNA conformation
f. Voltage applied
g. Presence of ethidium bromide
h. Concentration of the electrophoresis buffer