Exp No: 07

Pranshu Kumar
21BCB0134

AGAROSE GEL ELECTROPHORESIS OF NUCLEIC ACIDS

Objective:
To develop a basic understanding of electrophoretic theory, and to gain “hands-on” familiarity
with the procedures involved in horizontal gel electrophoresis to separate different molecules (ex:
DNA).

Introduction:
Electrophoresis is a technique that separates large molecules by size using an applied
electrical field and a sieving matrix. DNA, RNA and proteins are the molecules most often studied
with this technique; agarose and acrylamide gels are the two most common sieves. The molecules
to be separated enter the matrix through a well at one end and are pulled through the matrix
when a current is applied across it. The larger molecules get entwined in the matrix and retarded;
the smaller molecules wind through the matrix more easily and travel further from the well.
Molecules of the same size and charge migrate the same distance from the well and collect into a
band.
Agarose gel electrophoresis is widely used to separate molecules based upon charge, size
and shape. This is an analytical procedure used in several areas of Biotechnology, such as in
laboratories of investigation, biomedical and forensic fields. It is particularly useful in separating
charged biomolecules such as DNA, RNA and proteins. It is the easiest and most popular way of
separating and analyzing DNA (0.5- to 25-kb). Here DNA molecules are separated on the basis of
charge by applying an electric field to the electrophoretic apparatus. Shorter molecules migrate
more easily and move faster than longer molecules through the pores of the gel and this process is
called sieving. The gel might be used to look at the DNA in order to quantify it or to isolate a
particular band. The DNA can be visualized in the gel by the addition of ethidium bromide.
Agarose is a polysaccharide obtained from the red algae Porphyra umbilicalis. Its
systematic name is (1 4)-3, 6-anhydro-a-L-galactopyranosyl-(1 3)-β-D-galactopyranan. Agarose
makes an inert matrix. Most agarose gels are made between 0.7% and 2% of agarose. A 0.7% gel
will show good separation for large DNA fragments (5-10kb) and a 2% gel will show good
resolution for small fragments with size range of 0.2-1kb. Low percentage gels are very weak
(Note:- They may break when you lift them) but high percentage gels are usually brittle and do not
set evenly. The volume of agarose required for a minigel preparation is around 30-50ml and for a
larger gel, it is around 250ml.

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Structure of agarose (D-galactose and 3,6-anhydro-L-galactopyranose)

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 The gel is made by dissolving agarose powder in boiling buffer solution. The solution is then
cooled to approximately 55°C and poured into a gel tray where it solidifies. The tray is submerged
in a buffer-filled electrophoresis apparatus which contains electrodes.
Samples are prepared for electrophoresis by mixing them with components that will give
the mixture density, such as glycerol or sucrose. This makes the samples denser than the
electrophoresis buffer. These samples can be loaded with a micropipette or transfer pipette into
wells that were created in the gel by a template during casting. The dense samples sink through
the buffer and remain in the wells. A direct current power supply is connected to the
electrophoresis apparatus and current is applied. Charged molecules in the sample enter the gel
through the walls of the wells. Molecules having a net negative charge migrate towards the
positive electrode (anode) while net positively charged molecules migrate towards the negative
electrode (cathode). Within a range, the higher the applied voltage, the faster the samples
migrate. The buffer serves as a conductor of electricity and to control the pH. The pH is important
to the charge and stability of biological molecules.
The agarose gel consists of microscopic pores that act as a molecular sieve which separates
molecules based upon charge, size and shape. These characteristics, together with buffer
conditions, gel concentrations and voltage, affect the mobility of molecules in gels. Smaller
molecules move through the pores faster than larger ones. Molecules can have the same
molecular weight and charge but different shapes, as in the case of plasmid DNAs. Molecules
having a more compact shape (a sphere is more compact than a rod) can move more easily
through the pores. This means that the smaller the linear fragment, the faster it migrates through
the gel.

Figure: Agarose gel setup for agarose gel electrophoresis.

Ethidium Bromide is a fluorescent dye that is commonly added to agarose gels. This dye
intercalates between the bases of DNA, allowing DNA fragments to be visualized in the gel under
UV light and photographed. The intensity of the band reflects the concentration of molecules that
size, although there are upper and lower limits to the sensitivity of dyes. Because of its interaction
with DNA, ethidium bromide is a powerful mutagen and will interact with the DNA in your body
just as it does with any DNA on a gel. You should always handle all gels and gel equipment with

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gloves. Agarose gels with Ethidium Bromide must be disposed as hazardous waste in the labelled
container in the fume hood.
EQUIPMENTS AND MATERIALS REQUIRED
1. Electrophoresis chamber and power supply.
2. Gel casting trays, which are available in a variety of sizes and composed of UV-transparent
plastic.
3. Sample combs, around which molten agarose is poured to form sample wells in the gel.
4. Microwave oven and boiling water bath or steamer.
5. UV transilluminator (300 nm) Transilluminator (an ultraviolet light box), which is used to
visualize ethidium bromide-stained DNA in gels.
Note: Always wear protective eyewear when observing DNA on a Transilluminator to prevent
damage to the eyes from UV light
6. Polaroid camera or gel documentation system.

REAGENTS REQUIRED:
1. Agarose (molecular biology grade)
2. Electrophoresis buffer (Running buffer): usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA
(TBE).
To prepare stock solutions:
1OX TBE buffer: 545 g Tris, 278 g boric acid, 46.5 g EDTA in 5 L of sterile distilled water.
50X TAE buffer: 242 g Tris, 57.1 mL glacial acetic acid 100mL, 0.5M EDTA, pH8 .0, in 1 L of
sterile distilled water.
Prepare a 50x stock solution of TAE buffer in 1000 ml of distilled H2O:
 For this weigh 242 g of Tris base in a chemical balance. Transfer this to a 1000ml beaker.
 Prepare EDTA solution (pH 8.0, 0.5M) by weighing 9.31g of EDTA and dissolve it in 40ml
distilled water. EDTA is insoluble and it can be made soluble by adding sodium hydroxide
pellets. Check the pH using pH meter. Make the solution 100ml by adding distilled water.
 Pipette out 57.1 ml of glacial acetic acid.
 Mix the Tris base, EDTA solution and glacial acetic acid and add distilled water to make the
volume to 1000ml
Prepare sufficient electrophoresis buffer (usually 1X TAE ) to fill the electrophoresis tank and
to cast the gel:
 For this we take 2ml of TAE stock solution in an Erlenmeyer flask and make the volume to
100ml by adding 98ml of distilled water. The 1X working solution is 40 mM Tris-acetate/1
mM EDTA.
 It is important to use the same batch of electrophoresis buffer in both the electrophoresis
tank and the gel preparation.
3. Sterile distilled water.

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4. Gel loading buffer, which contains something dense (e.g. ficoll, sucrose or glycerol) to allow
the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the
gel and allow visual monitoring of the progress of electrophoresis.
Loading buffer: 50% (v/v) glycerol, 50 mM EDTA, pH 8.0, 0.125% (w/v) bromophenol blue,
1.125% (w/v) xylene cyanol.
To prepare a 6 X gel loading buffer containing glycerol and bromophenol blue, add 3 ml
glycerol (30 %), 25 mg bromophenol blue (0.25 %) and 10 ml of milli-Q, store at 4 °C. The
working concentration of the loading buffer should be 1 X.
5. Molecular weight size marker:
A molecular weight size marker is also called a DNA ladder is a set of standards that is used to
determine the approximate size of a test molecule during agarose gel electrophoresis.
DNA ladders are prepared by two ways: partial ligation, where a 100 bp of DNA piece is
partially ligated that gives rise to dimers of 200 bp, trimmers of 300 bp, tetramers of 400 bp,
pentamers 500 bp and so on. Secondly, restriction digestion of a known DNA sequence by a
particular restriction enzyme that gives rise to DNA pieces of varied molecular masses.
6. Ethidium bromide, a fluorescent dye used for staining nucleic acids.
Ethidium bromide: 10 mg/mL dissolved in H20. Store at 4°C in a container wrapped in tin foil.
(For the preparation of ethidium bromide adds 1 g of ethidium bromide to 100 ml of H2O. Stir
on a magnetic stirrer for several hours to ensure that the dye has dissolved. Wrap the
container in aluminum foil or transfer the 10 mg/ml solution to a dark bottle and store at room
temperature.)
7. DNA Sample:
5 μl of amplified product is sufficient to be visualized after agarose gel electrophoresis. It can
be mixed with 1 μl of 6 × gel loading dye to be loaded in the wells prepared in agarose gel.

PROCEDURE:
1. Prepare a solution of agarose in electrophoresis buffer at an appropriate concentration:
 Preparing Agaroge gel:
 For this usually 2 grams of agarose is added to 100ml of electrophoresis buffer.
Table: Agarose Gel Percentage and Efficient Range of Separation:

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2. Loosely plug the neck of the Erlenmeyer flask. Heat the slurry in a boiling-water bath or a
microwave oven until the agarose dissolves. The agarose solution can boil over very easily so
keep checking it. It is good to stop it after 45 seconds and give it a swirl. You can use a Bunsen
burner instead of a microwave - just remember to keep watching it.
3. Use insulated gloves or tongs to transfer the flask/bottle into a water bath at 55°C. When the
molten gel has cooled, add 0.5µg/ml of ethidium bromide. Mix the gel solution thoroughly by
gentle swirling.
4. While the agarose solution is cooling, choose an appropriate comb for forming the sample
slots in the gel.
5. Pour the warm agarose solution into the mold (gel tray) where it solidifies.
(The gel should be between 3 - 5 mm thick. Check that no air bubbles are under or between the
teeth of the comb.)
6. Allow the gel to set completely (30-45 minutes at room temperature), then pour a small
amount of electrophoresis buffer on the top of the gel, and carefully remove the comb. Pour
off the electrophoresis buffer. Mount the gel in the electrophoresis tank.
7. Add just enough electrophoresis buffers to cover the gel to a depth of approximately 1mm.
 Loading the gel
8. Mix the samples of DNA with 0.20 volumes of the desired 6X gel-loading buffer.
9. Slowly load the sample mixture into the slots of the submerged gel using a disposable
micropipette or an automatic micropipettor or a drawn-out Pasteur pipette or a glass capillary
tube. Load size standards into slots on both the right and left sides of the gel.
 Running the gel
10. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward
the positive anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between
the positive and negative electrodes). If the electrodes are 10cm apart then run the gel at 50V.
It is fine to run the gel slower than this but do not run it any faster. Above 5V/cm the agarose
may heat up and begin to melt with disastrous effects on your gel's resolution. If the leads
have been attached correctly, bubbles should be generated at the anode and cathode.
11. Run the gel until the bromophenol blue and xylenecyanol FF have migrated an appropriate
distance through the gel.
(The presence of ethidium bromide allows the gel to be examined by UV illumination at any
stage during electrophoresis).
 Gel staining and visualization
12. The gel tray may be removed and placed directly on a transilluminator. When the UV is
switched on we can see orange bands of DNA and measure their migration distance in mm.
Note these values in your lab book.
13. Photograph the gel on a UV trans illuminator.

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PRECAUTIONS
 Failure to dilute the TAE will result in very slow migration of the samples and very high
amperage.
 Ethidium bromide stains DNA by intercalating between the bases of DNA. It will also
intercalate into human DNA, so wear gloves to prevent contact with it.
 Make sure that the comb is nearest to the black electrode (cathode), as the DNA migrates
towards the red electrode (anode).
 Do not pull the comb out too soon before solidifying, as it causes the wells to collapse. It will
take 15 to 20 minutes to gel to solidify.
 Check the gel while it is running to make sure it is not getting too hot, as this will distort the
bands or melt the agarose.
 Wear UV protective glasses or cover the light box with the UV protective shield when the
UV light is on (during Gel visualization). The UV can cause skin cancer.

OBSERVATION:
Document the DNA on agarose gel by visualising under UV light. Match the banding position with
the corresponding position of the DNA ladder to get the approximate size of
the amplified product.

TROUBLESHOOTINGS

Problem Solution(s)
Gel doesn’t solidify 1) Not enough agarose added.
2) Not heated enough to dissolve the agarose.
Collapsed wells Gel wasn’t cool when the comb was removed
Skinny gel or irregular gel Agarose leaked past the dams. Put the dams in securely.
Well too small (sample won’t fit in the well) Agarose leaked past the dams. Put the dams in securely.
Gel runs too slowly 50X TAE not diluted correctly.
Gel runs too quickly Little or no 50X TAE added to electrophoresis buffer.
Only the ladder is visible under UV light 1) Poor DNA purification (e.g., minipreps).
2) Sample leaked out of the well.
No bands appear under UV light 1) No ethidium bromide was added.
2) Samples leaked out of the well
Fuzzy bands 1) Agarose wasn’t dissolved completely
2) Too much DNA
3) Particulates in the sample

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Results:

References:
 The Nucleic Acid Protocols Handbook, Ralph Rapley (Editors), Humana Press, 2000.
 Microbial Biotechnology- A Laboratory Manual for Bacterial Systems, by Surajit Das and
Hirak Ranjan Dash, Springer; 2015 edition.
 http://vlab.amrita.edu/?sub=3&brch=77&sim=1375&cnt=1.
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