Experiment No.

- 6
Agarose Gel Electrophoresis
Key points to be covered

1. What is gel electrophoresis? – Principle and Application

2. How agarose gel electrophoresis is different from PAGE?
3. Role of Ethidium bromide dye and TAE buffer
4. Role of loading dye and molecular marker
5. Observation of DNA under UV
 What is Gel electrophoresis?

• Gel electrophoresis is a technique commonly used in laboratories

to separate charged molecules like DNA, RNA and proteins
according to their size.
• Charged molecules move through a gel when an electric current
is passed across it.
• DNA is negatively charged, therefore, when an electric current is
applied to the gel, DNA will migrate towards the positively
charged electrode.
 How electrophoresis works?

• An electric current is applied across the gel so that one end of the
gel has a positive charge and the other end has a negative charge.
• The gel consists of a permeable matrix, a bit like a sieve, through
which molecules can travel when an electric current is passed
across it.
• Smaller molecules migrate through the gel more quickly and
therefore travel further than larger fragments that migrate more
slowly and therefore will travel a shorter distance. As a result the
molecules are separated by size.
 How agarose gel electrophoresis is
different from PAGE

• First discuss about the agarose gel electrophoresis and polyacrylamide gel
electrophoresis (PAGE).
o Agarose (A complex sugar material, generally extracted from seaweed. It is
frequently used for the separation of DNA by size using electrophoresis) gel is
used for larger molecules. This gel does not identify small differences between two
molecular bands. It is also solely used for the separation of DNA.
o Polyacrylamide gel is used for smaller DNA molecules. This gel identifies the
differences between molecular bands. Other than DNA, it is also used to separate
proteins.

• The higher the agarose concentration, the denser the matrix and vice versa. Smaller
fragments of DNA are separated on higher concentrations of agarose whilst larger
molecules require a lower concentration of agarose.
TAE Buffer
50X Stock – 2 M Tris, 1 M glacial acetic acid, 50 mM EDTA, pH – 8.3
1X working solution - 40 mM Tris, 20 mM glacial acetic acid, 1 mM EDTA,
pH – 8.3
Tris Acetate buffer, EDTA – sequester divalent cations (pH 8.0)
TAE (Tris-acetate-EDTA) buffer is used as both a running buffer and in
preparation of agarose gel.
Mention the role of EDTA ( why sequestration of divalent cations is important)
and importance of pH 8.3 for stability of DNA.
Importance – Buffer will provide ions for conductivity and maintain pH on
which DNA is stable.
TAE has a lower buffer capacity than TBE and can easily become exhausted,
but linear, double stranded DNA runs faster in TAE.
pH of tris buffer is temperature sensitive, please bring it to normal room
temperature before use.

• Note: Naresh Ji will fill the gel tank with buffer at the start of the day,
which can be used twice.
Loading Dye: (mention role of each component)
10 mM Tris-HCl (pH 7.6)
0.03% bromophenol blue,
0.03% xylene cyanol FF,
0.15% orange G
60% glycerol
60 mM EDTA.
Importance – to settle down DNA in wells and track to migration of DNA
In 1% agarose gels bromophenol blue co-migrates with ~300 bp DNA,
while xylene cyanol FF co-migrates with ~4000 bp DNA. Orange G co-
migrates with smallest fragments, i.e. ~50 bp.

DNA Ladder :
SM0331 Thermoscientic DNA ruler
0.5 microgram / gel will be used.
Importance : To know size of Isolated DNA.
Ethidium bromide staining

• Ethidium bromide is a molecule used to visualize DNA & RNA in agarose gel
electrophoresis experiments.
• DNA interchelator, inserting itself into the spaces between the base pairs of
the double helix. Ethidium bromide possesses UV absorbance maxima at 300
and 360 nm. Ethidium re-emits this energy as yellow/orange light centered at
590 nm. The fluorescence of ethidium bromide in aqueous solution is
significantly lower than that of the interchelated dye.
• ethidium bromide is that it is a potent mutagen. Solutions must be handled
with extreme caution, and decontaminated prior to disposal.
• More sensitive for Double stranded nucleic acids than single stranded
ones.
• Staining can be done in two ways
(i) Including Ethidium Bromide in the Gel Buffer
(ii) post run staining
Preparation of gel

Agarose gel
• Need to prepare 1 % agarose gel (0.4 gram in 40 ml of 1X TAE
buffer)
• Boil it in microwave oven until agarose get dissolved completely.
• Bring down the temp. and Add 2 µl of EtBr (10 mg mL-1) in 40 ml
gel preparation, be careful aout EtBr fumes.
• Put comb.
• Allow it to solidify.

Explain electrodes in electrophoresis tank.

Measure the distance between electrodes and explain why Gel is run at 70 -80 mV
(applied voltage should be 1-5V/cm)
Discuss why wells ( with DNA ) should be kept towards cathode while running the gel
and what will happen if opposite is done.
Thanks