This document describes the process of agarose gel electrophoresis. Key components include an electrophoresis tank, agarose gel mold, sample comb, electrophoresis buffers like TAE or TBE, loading buffer, ethidium bromide nucleic acid stain, and UV transilluminator. The DNA fragments migrate toward the anode based on factors like agarose concentration, voltage, and buffer composition. Higher agarose concentration or voltage separates smaller fragments, while different buffers affect migration speed. Ethidium bromide intercalates and allows visualizing DNA fragments under UV light.

1. Agarose Gel Electrophorises

2. Agarose Gel Electrophorises
• Electrophoresis tank and power pack
• The mold of the gel , available in various
sizes , made of transparent plastic with
UV .
• The open ends of the mold are sealed by
a strip while being poured and the gel
until it solidifies , then the strip is removed
prior to electrophoresis.
• The sample comb, around which the
molten agarose is poured to form the
wells of the gel in which the samples will
be filled.
• Buffer electrophoresis , most frequently
used are Tris - Acetate -EDTA ( TAE ) or
Tris-borate -EDTA ( TBE).

3. Agarose Gel Electrophoresis
• Loading buffer ( Loading buffer) ,
contains a dense material ( such as
glycerol ) to allow samples of ' down at
the bottom of wells.
• Ethidium bromide, is an agent
commonly used as intercalate nucleic
acid marker . NOTE : Ethidium
Bromide is a mutagenic and should be
treated as a dangerous chemical -
wear gloves during use.
• Transilluminator ( ultraviolet light
source ), which is used to visualize
Ethidium bromide intercalated into
DNA . NOTE : Always wear protective
glasses looking at DNA under UV light
to avoid damaging the eyes

4. Principles and Factors
• The DNA will move towards the
anode ( positive electrode ) which is
usually red and is dependent on
the;
– Agarose concentration
– Voltage electrophoresis buffer
– The effects of Ethidium bromide

5. Concentration of Agarose
• Increasing the concentration of agarose in
a gel facilitates the separation of DNA
fragments of smaller size. While less than
agarose concentration in the gel permits
the separation of the DNA fragments
larger

6. Voltage
• When the voltage applied to a gel increases ,
fragments of larger sizes migrate proportionately
faster than the small fragments . For this reason
the separation of fragments of size greater than
2K can be achieved by the application of not
more than 5 volts per cm for the gel (the value of
cm is the distance between the two electrodes
and not the length of the gel)

7. Electrophoresis Buffers
• Most often used to double-stranded DNA are: TAE
( Tris- Acetate - EDTA) TBE ( Tris- borate - EDTA).
• The DNA fragments will migrate at slightly different
speeds in both buffers because of the difference in
the ionic strength.
• Not only establish the pH buffers, but also they
provide support to the ion conductivity. If you
accidentally use water in place of the buffer there
will be no DNA migration on the gel. Also, if you use
a high concentration of buffer (eg 10 X solution) may
be enough heat generated to melt and even the
tray.

8. Effects of Ethidium bromide
• Ethidium bromide is a fluorescent
material which intercalates between
the bases of the nucleic acid and
allows convenient detection of DNA
fragments in gel.
• It can be included in the Agarose gel
or added to the DNA prior to
deposition on gel samples to allow
viewing of the migration of DNA
fragments on the gel.
• The binding of the Ethidium bromide
to the DNA changes its mass and its
rigidity therefore the mobility of the
DNA

9. The gel was exposed to ultraviolet radiation, The ethidium bromide stained
DNA in a red - orange color

10. Photography
1.Well molecular size marker
ladder ( 1kbplus )
2.Empty wells .
3.A well PCR product of
slightly greater than 500
base pairs in size .
4.Well Fragment of about
4.5kb

11. Tank filled with buffer
and transformer used
for Agarose gel
electrophoresis