AGAROSE GEL

ELECTROPHORESIS

SATYAJIT. S
1styr MSc Biotechnology
 OUTLINE OF CONTENTS
 Introduction to agarose.
 Agarose gel electrophoresis
 Principle
 Materials required
 Components in Detail
 Protocol
 Applications
 References.
 AGAROSE
Agarose is a highly purified, uncharged, linear polysaccharide whose monomer units are D-galactose and 3,6-anhydro L-
galactopyranose. Its molecular weight is around 120000 g/mol.
Agar is a cell wall and intercellular component of some red algae such as Gelidium and Gracillaria. Agarose is purified
from agar by the removal of agaropectin(Sulphate fraction).
Agarose is a free flowing white powder whose boiling point is close to that of water and it forms a gel around 37-43 ° C.
Gel point is the temperature at which the aqueous solution of agarose forms the gel. Agarose exhibits hysteresis in its
transitions(their gel point is not the same as their b.p). The hydroxyl groups present in the exterior help it to aggregate
into a matrix and is stabilized by water.
The agarose forms a mesh like structure in the gel and is
responsible for the pores. The pore size is related to the
concentration of agarose used.
Agarose gels are basically hydrocolloids which are held
together by weak hydrogen bonds and hydrophobic bonds.
Intermediate or combinations of std. and N-S gels are preferred to get sturdy gels with better resolution.
Most commonly used concentration ranges from 0.7-2% .
 AGAROSE GEL ELECTROPHORESIS
Electrophoresis is a technique used to separate macromolecules(proteins and nucleic acids)based on their size,shape
and charge which depends upon the mobility and migration of the molecules under the influence of an external
electric field.
Agarose gel electrophoresis is the most common, routine and preferred technique used to separate DNA molecules
based on their size only.

PRINCIPLE
The negatively charged DNA molecules migrate towards the positive end (Anode)under the influence of constant
current, hence the separation of molecules occurs based on the mass and charge. But since all the DNA fragments have
the same amount of charge per mass,the separation is purely based on the size of the individual fragments.
The separation is brought about by the sieving effect of the gel and its pores.
The DNA molecules are forced to move through the pores of the gel due to the electric field.
The negative charge of the phosphate backbone is responsible for imparting the negative charge which moves towards
the anode.
The migration rate depends upon the following factors-
• Agarose Concentration
Lower the concentration of agarose used, larger the pores. This makes large fragments move easily and faster.
• Conformation of the DNA
Supercoiled dna is more compact and hence it runs faster than circular or linear DNA.
• Size of the DNA fragment
Smaller molecules migrate more easily than longer molecules through the pores. Hence smaller fragments will be more
closer to the anode at the end while large fragments remain near the wells.
• Applied Voltage and Temperature
Higher the voltage, More quickly the gel runs. Increase in voltage inturn increases the temperature which would
increase migration. But if the voltage is too high, then the gel might melt and high temperatures will denature the
sample.
• Ionic strength of buffer
Higher the ionic strength of the buffer, it reduces the net charge of the molecules. Hence the molecules tend to move
slowly.
 MATERIALS REQUIRED
 Agarose Gel
 Gel Casting Trays
 Gel Documentation System
 Buffers (Includes Running, Loading and Gel buffer)
 DNA Ladder/Marker
 Ethidium Bromide
 Electrophoresis equipment (Includes power supply, Gel tank, Gel combs, Casting
trays, Electrodes and cover).
 COMPONENTS IN DETAIL
1) BUFFERS
Buffers are mainly used in gel electrophoresis to facilitate ions that carry current and provide the liquid medium for the
migration of DNA. It also maintains the pH within the desired range without much fluctuations. They also react with
products of electrolysis of water to maintain pH.
Commonly used buffers are TAE(Tris,Acetate,EDTA) or TBE(Tris,Borate,EDTA) in 1X Concentration. The overall pH is 8.3
Tris is the main component which maintains the buffer pH.
EDTA is a chelating agent that removes divalent and trivalent metal ions such as Mg2+ and Ca2+.This inactivates metal
dependent nucleases which would otherwise cleave the DNA or RNA.
Acetate and Borate are both acidic and help in bringing down the basicity of tris.

Composition of 10X TBE buffer (250ml) Composition of 10X TAE buffer (250ml)
Tris- 900mM 400mM Tris
Boric Acid- 890mM 200mM acetic acid
EDTA- 2mM 10mM EDTA
 Gel Buffer- The gel buffer consists of 1X TAE/TBE, Agarose and EtBr.
 Running/Tank Buffer- It is only 1X TAE/TBE.
 Loading Buffer- The loading buffer contains Bromophenol blue(BPB)/Methylene blue, Ficoll 400/Glycerol and Xylene
Cyanol.
BPB is a pH indicator and also it indicates the progression of migration. It has a negative charge hence moves towards
the anode along with the DNA. It moves faster than DNA and also does not react with any other components.
Xylene Cyanol is also a tracking dye that shows the status of progression which could be used in combination with BPB.
Ficoll 400 is a high mol.wt polysaccharide which helps in providing density to the buffer, hence the DNA samples stay
inside the wells.
Composition of 10X loading buffer
0.42 % (W/V) Bromophenol blue powder
25 % Ficoll
0.42% (W/V) Xylene cyanol FF (optional)

2) ETHIDIUM BROMIDE(EtBr)
EtBr is a fluorescent dye that intercalates between the bases of nucleic acids and allows easy detection. On exposure to UV
it fluorescences and gives an orange colour. After intercalating in between the base pairs it removes water and emits
fluorescence. The ring structure of ethidium is hydrophobic it resembles the rings of the bases. It binds to the bases
through van der waals interactions. Since the binding causes changes in conformation, flexibility and molecular weight of
DNA it is a very potent mutagen. When exposed to UV light, electrons in the aromatic ring of the ethidium molecule are
excited, which leads to the release of energy (light) as the electrons return to ground state. Usual Standard concentration is
0.5-1μg/ml.
3) DNA ladder
DNA ladder is a set of standard DNA fragments whose
molecular weights are known. The ladder is used to
calibrate the gel so that it can be compared with the
unknown samples. Since it contains regularly spaced
fragments it looks like a ladder on the gel. When it is run
alongside the other samples, the rough estimate of the
molecular weight can be identified.
 PROTOCOL
1) PREPARATION OF AGAROSE GEL
Add agarose powder in a flask according to the concentration requirement. Add required volume of 1X TAE/TBE
buffer and mix well. Heat the mixture in an oven until all the agarose is dissolved(Solution becomes clear).
Cool down the solution and add 20μl of ethidium bromide(0.2-0.5 μg/ml).Mix this solution gently.
2) CASTING OF THE GEL
Place the gel tray(UV transparent available) on the gel caster and tighten the clamps. Don’t forget to place the combs
before pouring the gel onto the gel tray and allow it to solidify which takes about 30 mins.
Meanwhile fill the electrophoresis chamber with the tank buffer and also submerge the gel completely with the same
buffer. Make sure that the wells are towards the Cathode end.
3) LOADING OF SAMPLES
Carefully remove the comb from the gel. This should form the wells for our samples.
Place the DNA samples onto an Al foil or parafilm and mix them with 10-20 μl of the loading buffer.
Carefully add 10-20μl of the mixed sample into the wells without breaking the gel. Ensure that the entire sample
has gone into the well. The DNA ladder can be added in the 1st or last well.
4) RUNNING THE GEL/ELECTROPHORESIS
Close the tank with its cover and connect the electrodes to the power supply. The gel is run at 80-100V for around
an hour. The samples are run until they reach around 75% of the distance from the well. The loading dye would run
faster than the samples hence switch off the power supply when the dye has almost reached the end of the gel.
5) VISUALISING THE GEL
After switching off the power supply, remove the gel from the chamber and drain off any excess buffer. Then place
the gel either in an UV transilluminator or a gel documentation system. In both the cases UV light is used as the
source. EtBr intercalates to the hydrophobic interior of the DNA. When exposed to UV it fluorescens and hence we
can see the presence of orange DNA bands. In a gel doc, a digital camera is fitted to take the pictures.
 APPLICATIONS
 Estimation of size of DNA molecules after restriction digestion.
 To detect the presence of nucleic acids following extraction.
 Analysis of PCR products from DNA fingerprinting, molecular diagnosis, RFLP and VNTR analysis.
 An alternative method to separate a mixture of DNA.
 Segregation of DNA and RNA in terms of length of the fragments.
 Study of DNA topology.
 Seperation of DNA fragments for further downstreaming.
 REFERENCES
 J Vis Exp. 2012 Apr 20;(62). pii: 3923. doi: 10.3791/3923
 Smith D.R. (1993) Agarose Gel Electrophoresis. In: Murphy D., Carter D.A. (eds) Transgenesis Techniques.
Methods in Molecular Biology™, vol 18. Humana Press
 geneticeducation.co.in
 khanacademy.org
 slideshare.net
 sciencedirect.com
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