PRACTICAL REPORT

On

Preparation of Agarose gel and study the discriminatory power of Agarose using
different % of Agarose.

Submitted by

Thevar Jayabharathi.P

Msc.BT-18016
INTRODUCTION
Agarose gel electrophoresis is a method of gel electrophoresis used
in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed
population of macromolecules such as DNA or proteins in a matrix of agarose.Agarose is
a polysaccharide, generally extracted from certain red seaweed.It is a linear polymer made up
of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-
anhydro-L-galactopyranose.Agarose is one of the two principal components of agar, and is
purified from agar by removing agar's other component, agaropectin. For agarose, the gel
concentration is defined in g/100 ml (Hjertén, 1962) while the pore size of a polyacrylamide
gel can be controlled by the total amount of acrylamide present (%T, where T = total
concentration of acrylamide and bisacrylamide monomer) and the degree of cross-linking
(%C, where C = bisacrylamide concentration) (Rüchel, 1978). This procedure, in fact,
resembles a sieving process where the gels act as a sieving medium, defining nucleic acids
based on fragment size in response to an electric current. The gels can be casted in a variety
of shapes, sizes, and porosities. The choice within these parameters depends primarily on the
fragments to be separated. The sugar polymers that make up the agarose gel matrix
(powdered agarose heated in appropriate buffer, poured into a gel tray and allowed to
solidify) act like a sieve. The concentration of gel affects the resolution of DNA separation.In
principle,when a direct current is introduced,molecules and particles usually in aqueous
solution,will migrate towards the direction of electrode bearing the opposite charge.Since
they are varying in terms of masses and charges,different molecules and particles of a
mixture will migrate at different velocities.Basically the mobility of nucleic acid in gel is
influenced by factors such as gel concentrations,buffer conditions,sizes and
conformation.Agarose gel electrophoresis is the benchmark technique for separation of
nucleic acids.For a standard agarose gel electrophoresis, a 0.8% gives good separation or
resolution of large 5–10kb DNA fragments, while 2% gel gives good resolution for small
0.2–1kb fragments. 1% gels is often used for a standard electrophoresis.The concentration is
measured in weight of agarose over volume of buffer used (g/ml). High percentage gels are
often brittle and may not set evenly, while low percentage gels (0.1-0.2%) are fragile and not
easy to handle. Low-melting-point (LMP) agarose gels are also more fragile than normal
agarose gel. Low-melting point agarose may be used on its own or simultaneously with
standard agarose for the separation and isolation of DNA.[26] PFGE and FIGE are often done
with high percentage agarose gels.Agarose concentration plays an important role in
electrophoretic separation.The gels porosity is directly related to the concentration of agarose
in the medium and it determines the size range of DNA molecules that can be adequately
resolved. In a given size,a linear DNA fragment will migrate at different rates through gels
containing different concentrations of agarose. The greater the agarose concentration, the
smaller the pores created in the gel matrix, and the more difficult it is for large linear DNA
molecules to move through the matrix. Changing the agarose concentration changes the size
of the sieve matrix of the gel. However, there is an upper and lower limit to accurate
separation of DNA molecules using agarose gel electrophoresis. The rate of migration of a
DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2)
agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium
bromide, 6) type of agarose and 7) electrophoresis buffer.

MATERIALS REQUIRED:

1)Agarose powder

2)1XTAE buffer
3)DNA Sample
4)Ethidiumbromide
5)Electrophoresis tank
6)Geltray
7)Comb
8)Power supply
9)Weighing machine
10)Micro oven
11)DNA marker

METHODOLOGY
1. Preparation of the Gel

1. Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are
prepared using a w/v percentage solution. The concentration of agarose in a gel will
depend on the sizes of the DNA fragments to be separated, with most gels ranging
between 0.5%-2%. The volume of the buffer should not be greater than 1/3 of the
capacity of the flask.
2. Add running buffer to the agarose-containing flask. Swirl to mix. The most common
gel running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM
Tris-borate, 1 mM EDTA).
3. Melt the agarose/buffer mixture. This is most commonly done by heating in a
microwave, but can also be done over a Bunsen flame. At 30 s intervals, remove the
flask and swirl the contents to mix well. Repeat until the agarose has completely
dissolved.
4. Add ethidium bromide (EtBr) to a concentration of 0.5 μg/ml. Alternatively, the gel
may also be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr
for 15-30 min, followed by destaining in running buffer for an equal length of time.

Note: EtBr is a suspected carcinogen and must be properly disposed of per institution
regulations. Gloves should always be worn when handling gels containing EtBr. Alternative
dyes for the staining of DNA are available; however EtBr remains the most popular one due
to its sensitivity and cost.

1. Allow the agarose to cool either on the benchtop or by incubation in a 65 °C water

bath. Failure to do so will warp the gel tray.
 2. Place the gel tray into the casting apparatus. Alternatively, one may also tape the open
edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to
create the wells.
3. Pour the molten agarose into the gel mold. Allow the agarose to set at room
temperature. Remove the comb and place the gel in the gel box. Alternatively, the gel
can also be wrapped in plastic wrap and stored at 4 °C until use.

MAKING 1% agarose gel:

 Weigh 0.5g of agarose and dissolve it in 50ml of1XTAE buffer.
 Heat the solution over a hot plate to boiling consistency marked
with a clear solution.
 Leave the solution to cooland add 2ul of EtBr solution mix it well
by gentle swirling.
 Pour it in the gel tray-comb setup.Also besure the gelplates has
been taped securely and contain well combs prior topouring.
 Allow the solution to cool and harden to form gel.
 Prepare similarly for 1% and 1.5%agarose gel

2. Setting up of Gel Apparatus and Separation of DNA Fragments

1. Add loading dye to the DNA samples to be separated . Gel loading dye is typically
made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30%
glycerol). Loading dye helps to track how far your DNA sample has traveled, and also
allows the sample to sink into the gel.
2. Program the power supply to desired voltage (1-5V/cm between electrodes).
3. Add enough running buffer to cover the surface of the gel. It is important to use the
same running buffer as the one used to prepare the gel.
4. Attach the leads of the gel box to the power supply. Turn on the power supply and
verify that both gel box and power supply are working.
5. Remove the lid. Slowly and carefully load the DNA sample(s) into the gel . An
appropriate DNA size marker should always be loaded along with experimental
samples.
6. Replace the lid to the gel box. The cathode (black leads) should be closer the wells
than the anode (red leads). Double check that the electrodes are plugged into the
correct slots in the power supply.
7. Turn on the power. Run the gel until the dye has migrated to an appropriate distance.

3. Observing Separated DNA fragments

1. When electrophoresis has completed, turn off the power supply and remove the lid
of the gel box.
2. Remove gel from the gel box. Drain off excess buffer from the surface of the gel.
Place the gel tray on paper towels to absorb any extra running buffer.
3. Remove the gel from the gel tray and expose the gel to uv light. This is most
commonly done using a gel documentation system . DNA bands should show up as
orange fluorescent bands. Take a picture of the gel.
 4. Properly dispose of the gel and running buffer per institution regulations.

RESULT AND DISCUSSION

The migration distances of DNA fragments undergoing gel electrophoresis were compared, in
order to study the separation capacity of gels with different agarose concentrations. Different
concentration of Agarose gel were prepared and DNA was run in all the different
concentration gel.

For the BACTERIAL(E.coli) DNA 1%separation was good then 0.8% and 1.5%.
 As seen from the result, some bands which were thick and intense werenot seen in
0.8% concentration because at this lower concentration higher fragments were able to
move efficiently and smaller fragments movement was retarded (in 2nd lane).

 In comparison to 1% and 1.5%, 1.5% gel only allowed the smaller fragments
separation and larger fragments were not efficiently separated.

For the PLANT GENOMIC DNA

 1%agarose gel gave good separation of bands in all the lanes as compared to
1.5%agarose gel in which the last well 9th lane some bands were not visible due to the
inability of larger fragment DNA to migrate in higher concentration.

DISCUSSION & CONCLUSION:

It will become increasingly important to be able to choose and carry out the appropriate
electrophoresis technique for specific separation problems.
For DNA fragments of 4-23kb a low agarose concentration has a higher separation capacity.
The fragments between 2 and 0.5kb can be separated better in gels with higher agarose
concentration . Since the fragment length range from 2-4kb is equally well separated in 0.8%
and 1% agarose gels, 0.8% Agarose concentration is suggested
By applying two different Agarose concentrations, electrophoresis can be conducted rapidly
with a higher voltage while providing a better resolution of DNA fragments. The only
drawback of this suggested technique is perhaps the method of preparing a gel with two
agarose concentrations which might be slightly time-consuming and tedious as compared to
the traditional method of gel preparation.
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