Experiment :5

Agarose Gel Electrophoresis

 Key points
1. What is gel electrophoresis?
2. How does electrophoresis works?
3. How agarose gel electrophoresis is different from PAGE?
4. Role of :
 Ethidium bromide
 TAE buffer
 Loading dye
 DNA ladder
5. Preparation of agarose gel
6. Analyzing the gel.
 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 or charge.

• 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.
DNA Gel electrophoresis VS PAGE ?

Two types of gel matrix used for electrophoresis:

o Agarose gel (Agarose is polysaccharide extracted from Red

algae) is frequently used for the separation of larger molecules
like DNA on the basis of size. This gel does not identify small
differences between two molecular bands. Agarose
concentration is uniform throughout

o Polyacrylamide gel- is used for smaller molecules mainly RNA

and proteins. This gel identifies the small differences between
molecular bands.
 When to use either technique: Horizontal gel electrophoresis is mainly
used in separating mixtures containing DNA and RNA molecules while
vertical gel electrophoresis is ideally used in separating Small RNAs &
proteins.
 Gel electrophoresis can be conducted in either a horizontal or vertical orientation.
Horizontal gel electrophoresis
1. Orientation and Buffer System: gel matrix
(agarose) is casted horizontally and submerged
in a continuous running buffer.
2. The system is divided into two compartments,
with agarose gel separating the two with an anode
located at one end, while a cathode at the other.
3. The compartments are exposed to atmospheric
oxygen.
Vertical gel electrophoresis
1. The gel( acrylamide) is vertically oriented and
the buffer system may be continuous (Native
PAGE) or discontinuous (SDS- PAGE)
2. The system is more complex. A thin gel (<2
mm) is poured between two glass plates and
mounted so that the bottom of the gel is
submerged in buffer in one chamber and the top
is submerged in buffer in another chamber
3. The exposure of compartments to atmospheric
oxygen is prevented in this method since oxygen
inhibits the polymerization of acrylamide, and
thus, interferes with the creation of the gel. So
acrylamide cannot be used for horizontal systems
Role of Ethidium bromide dye and TAE buffer
Ethidium bromide
• Ethidium bromide is a molecule commonly used to visualize DNA in agarose gel
electrophoresis experiments.
• It binds to DNA and fluoresces under the proper conditions (UV light) and known to be
an intercalating agent.
• The flat structure of ethidium bromide allows it to intercalate, or insert, between
nitrogenous bases of a DNA molecule.
• Ethidium bromide is that it is a potent mutagen. Solutions must be handled with
extreme caution and decontaminated prior to disposal.

TAE buffer
• TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.
pH 8.3 (mention importance of pH )
• Tris and Acetic acid- maintaining the pH and buffering capacity.
• EDTA- chelates the divalent cations
• Buffer provides ions for conductivity and maintain pH so that DNA remains stable. It is
used as both a running buffer and in preparation of agarose gel. Avoids heating and
melting of gel.
Role of Ethidium bromide
 Role of loading dye and DNA Ladder
Loading Dye
• Loading dye is added to DNA sample to give it color to the naked eyes.
• Helps with gel loading and allows you to gauge how far the DNA has migrated;
• Contains:
1. 0.25% (w/v) Bromophenol blue- Tracking dye to monitor progress of gel
2. 0.25% Xylene cyanol- tracking dye used in combination with bromophenol blue
to increase its effectiveness and quality of result.
3. 0.1% methylene blue
4. 30% Glycerol- increases the density of your DNA sample so that it can settle to
the bottom of the gel well, instead of diffusing in the buffer.
DNA ladder
• A molecular-weight size marker, is a set of standards that are used to identify the
approximate size of a molecule run on a gel during electrophoresis.
• It contains DNA fragments of known length, making them suitable for
estimating the fragment length of concurrently run samples.
• Available as 50bp, 100bp, 1000bp(1Kb) or 3000bp
Preparation of Agarose gel
• Prepare 1 % agarose gel (weigh 0.4 gram and add in 40 ml of 1X TAE
buffer)
• Boil/ microwave in oven for 1-3 mins until agarose get dissolved
completely.
• Allow agarose solution to cool down(when you can comfortably keep
your hand on the flask) and Add 2 µl of EtBr (10 mg mL-1) in 40 ml gel
preparation. Be careful with the EtBr fumes.
• Pour the agarose into a gel tray with the well comb in place. Allow it to
solidify.

Concentration of agarose- agarose gel matrix acts like a sieve. % of

agarose determines size of the sieve matrix. The greater the percentage of
agarose, the smaller the linear DNA that can be resolved. Larger molecules
can be resolved with lower concentration of agarose.
Loading samples and Running on Agarose Gel
Once solidified, place the agarose
gel into the electrophoresis unit
filled with TAE Buffer.
Add loading dye to each of your
DNA samples.
Carefully load the DNA ladder into
the first well and samples into the
additional wells of the gel (prevent
bubbles or buffer from entering the
tip)
Run the gel at 80-100 V until the
dye line is approximately 75-80% of
the way down the gel.
A typical run time is about 1-1.5
hours, depending on the gel
concentration and voltage.
Movement of DNA is always from negatively charged cathode
(Black) towards positively charged anode (Red)
 Analysing the Gel
 Using the DNA ladder in the first lane as a guide you can infer the size of the DNA in your
sample wells.

ETBR stained gel

Results
Applications of gel electrophoresis

•In the separation of DNA fragments for DNA fingerprinting to

investigate crime scenes.

•To analyze results of polymerase chain reaction.

•To analyze genes associated with a particular illness.

•In DNA profiling for taxonomy studies to distinguish different

species.