WHAT I S GEL ELECTROPHORESI S?

Gel electrophoresis is a lab procedure by which one can separate DNA fragments*, according to
their size and charge, in a sample, using the principles of electrolysis. Electricity is used to aid
the movement of negatively charged DNA fragments from the cathode to the anode, through a
gel matrix.
*RNA and proteins can also be separated
APPARATUS:
1) DNA sample, which is to be tested
2) DNA restriction ladder, to use as a means of calibration for measuring the size of DNA
fragment
3) Micro-pipettes, to extract and release DNA samples and dye onto the wells of the agarose
gel
4) Agarose gel, to observe the relative speeds of the DNA fragments and hence, make an
estimation of the size of each fragment
5) Power supply, to generate electricity for carrying out electrophoresis
6) Comb to make equi-spaced wells of the same size in the agarose gel
7) Spacer, to make sure that the comb does not make a hole in the gel
8) Ethidium bromide solution, which is a fluorescent material
9) Gel box, apparatus in which electrophoresis will be carried out
10) Loading dye, to be mixed with every DNA sample in order to track their movements
11) UV light box, to fluoresce the DNA fragments intercalated with ethidium bromide
12) Camera, to take picture for further records
13) Running buffer solution, which completely fills the gel box to provide ions to the sample
for more conductivity and maintain a constant pH
14) Loaded buffer, to make the DNA samples heavy
15) Restriction enzymes, to break given DNA into smaller fragments
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Figure: Gel electrophoresis apparatus set-up
PROCEDURE:
1) The DNA samples are mixed with desired restriction enzyme, loading dye and buffer.
The restriction enzyme breaks the DNA sample into fragments. The buffer contains
glycerol which will make the DNA samples heavy causing them to sink to the bottom of
the wells, so that the samples do not flow out of the wells and dye, which binds to the
DNA molecules, helps to track the movement of the DNA fragments as it travels slightly
faster than the fragments, therefore helping in understanding when to turn the power
supply preventing all the fragments from reaching the anode.
2) The comb is first placed near the end of the of the gel box to which the negative electode
will be connected. A spacer is initially placed underneath the comb to ensure that the
depth of the comb is not low enough to penetrate through the gel and produce holes (this
would cause the DNA fragments to sink underneath the gel).
3) The spacer is removed after the required depth is obtained.
4) The leveler of the box is turned on to make sure that the gel has a flat surface.
5) Hot agarose solution, which has been heated in a microwave beforehand, containing
ethidium bromide is then poured into the gel box from a conical flask, making sure that
the solution does not exceed the pour line. The ethidium bromide will bind to the DNA
fragments and will fluoresce near UV light.
6) The solution is left to stand until it solidifies into a gel.
7) The comb is removed from the gel.
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8) The gel box is then filled with running buffer solution, making sure that the gel and the
wells are submerged with it. The buffer solution adds ions to the solution to increase
electrical conductivity and also helps to maintain a constant pH.
9) The first well is filled with a standard DNA ladder or DNA marker, which will be used as
an arbitrary means of calibration to measure the size of every DNA fragment.
10) The micropipette is used to transfer the DNA samples to the other wells accordingly.
11) The power supply is turned on. The DNA fragments in the samples should migrate from
the negative end to the positive end with time, since the phosphate group makes DNA
negatively charged.
12) The buffer solution must show slight bubbling as a sign of electrical conductivity.
13) As soon as the dye reaches the positive electrode, switch the power supply off.
14) Observe the agarose gel under UV light.


Figure: A gel electrophoretogram under UV light


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OBSERVATI ONS AND I NTERPRETATI ONS:
There are two observations to be made from the sample-
1) The number of fragments in each DNA sample. This can be deduced by counting the
number of clearly distinguishable dark bands of concentrated DNA and dye, produced
from each well. This is because different fragments will be of different lengths and hence
will travel through the gel matrix at different speeds, resulting in separate distinguishable
bands of DNA.
2) The size of each DNA fragment from every sample. This can be measured by using the
standard DNA ladder which allows us to estimate the length of each DNA fragment, in
terms of number of base pairs. The size of any fragment from the DNA sample is equal to
that from the standard DNA ladder, if they correspond to each other. Whether the two
correspond to each other, can be found by looking at the electrophoretogram under UV
light, which will cause the EtBr bound with every DNA to fluoresce. The fragments
closest to the cathode will be of the largest size and those furthest away from the cathode
will be of the smallest size. This is because the agarose gel is a matrix with small spaces.
This means that DNA fragments which are smaller can move through this spaces faster
than larger DNA fragments and hence, can travel a faster distance than the larger
fragments over a particular time period.

Figure showing the apparatus for gel electrophoresis and the use of DNA markers
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Let us consider the results of gel electrophoresis shown above. There are three samples of DNA
and a DNA marker which is considered as the molecular weight standards. The arbitrary
measurements on the extreme right refer to the size of each DNA fragment.
Sample A had two DNA fragments one at 70 Kbp and the other at 30 Kbp; B had two as well
one at 60 Kbp and the other at 40 Kbp; and C had four fragments one at 40 Kbp, one at 30
Kbp, one at 20 Kbp and the other at 10 Kbp.
PRECAUTI ONS:
Experimental-
1) Handle the agarose gel with care and make sure it does not break during the procedure.
This may cause the DNA samples to leak out of the gel, nullifying the authenticity of the
results.
2) The wells must be made near the cathode since DNA is negatively charged.
3) We must not make a hole in the agarose gel when making holes. Otherwise, DNA sample
may leak out of the gel
4) Wait for the gel to completely solidify before pulling the comb out of the wells, or the
wells may collapse.
Safety-
1) Prevent skin and eye contact with ethidium bromide by wearing lab coat, putting on
gloves and eye goggles, since EtBr is a mutagen.
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2) Although low level of electricity is being used, we must take care when using the power
supply.
3) Excessive exposure to UV light must be minimized by wearing a lab coat lined with lead
near the UV light box.
4) Wear gloves when using dye as, if it falls on the skin it may cause stains which are
difficult to remove.
5) Take care with hot agarose solution after it has been heated in a microwave for
liquification.
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