Gel Electrophoresis

Turquoise_team
LAB NO.

4
13-17/12/2015
Final

Part one: Gel Electrophoresis

BACKGROUND
Agarose gel electrophoresis is the easiest and most popular used method to
separates molecules on the basis of electrical charge, size, and shape. The method is
useful in separating charged biologically important molecules such as DNA
(deoxyribonucleic acids), RNA (ribonucleic acids), and proteins. Agarose forms a
porous gel when boiled and cooled in a suitable buffer. When 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 DNA can be visualized in the gel by the addition of ethidium
bromide and exposed to UV light.

PRINCIPLE:
The agarose gel contains molecule sized pores. The pores in the gel control the speed
that molecules can move. Smaller molecules move through the pores more easily
than larger ones.

The gel setup provides wells for loading DNA in to it. The loaded negatively charged
DNA molecules (due to the presence of PO4- groups in their backbone structure)
move towards the positively charged electrode (anode) and get separated along the
length of the gel. The ethidium bromide that has intercalated into the DNA molecule
will absorb the UV light and fluoresce. The actual size of a DNA molecule can be
inferred by comparing the distance that an unknown DNA molecule moved with the
distance that known standards moved.

To monitor the DNA direction a

negatively charged loading dye
always used. The loading dye gives
colour to the sample to make it easy
to load into the wells. Loading dye
move in the same direction as the
DNA during electrophoresis. The gel
loading dye possesses bromophenol
blue and xylene cyanol. Xylene cyanol
gives a greenish blue colour while
bromophenol blue provides bluish
colored zone. The successful DNA run
is determined by the presence of
both the colored dye in the gel.
The dye also guide us to make sure that the DNA do not get out of gel.

FACTORS AFFECTING THE MOVEMENT OF DNA:

The size & charge
Smaller molecules will penetrate the pores faster than larger molecules. Regarding
that all DNA molecules will have negative charge, due to the phosphate backbone.
In addition we can know the size from the moving of DNA on gel by using marker
DNA or ladder ( known DNA sizes available commercially ).
Voltage Applied
The moving rate of the linear DNA fragments through agarose gel is proportional to
the voltage applied to the system. However, voltage should be limited because it may
melt the gel. ( the maximum voltage = about 160 v )
Ethidium Bromide(EtBr)
It is an intercalating agent which intercalates between nucleic acid bases and allows
the convenient detection of DNA fragments in gel. EtBr is a known "mutagen",
however, safer alternatives are available. Binding of Ethidium bromide to DNA alters
its mass and rigidity, and thereby its mobility. ( it is positively charged so it will affect
the DNA movement in the gel )
Buffers
buffers provide the ions for supporting conductivity( ). The most commonly used
buffers are Tris-acetate-EDTA (TAE) and Tris-borate-EDTA( TBE).
Confirmation of DNA
DNA with different conformations that has not been cut with a restriction enzyme will
migrate with different speeds. Nicked or open circular DNA will move slower than
linear and super coiled DNA. Super helical circular, nicked circular and linear DNAs

migrate gels at different rates through agarose gel. The relative nobilities of these
three forms depend on the concentration, type of agarose used to make the gel,
applied voltage, buffer, and the density of super helical twists.
Different concentrations of agarose are useful for isolating different-sized DNA
fragments.
% agarose
Size of fragments separated (kbp)
0.3
0.6
0.7
0.9
1.2
1.5
2.0

60 - 5
20 - 1
10 - 0.8
7 - 0.5
6 - 0.4
4 - 0.2
3 - 0.1

To separate different sizes of DNA we use

different concentrations of agarose gel.
For smaller sizes we use high concentration
of agarose, because it will prevent the small
DNA fragment from moving fast. However
for larger DNA we use low concentration to
allow the huge fragments to move.

Materials Required:
Electrophoresis buffer: 1x TBE buffer ( running buffer )
Agarose ultra pure (DNA graded)
electrophoresis tank, gel tray, sample comb and power supply
Plastic or insulation tape
Ethidium bromide: 10 mg /ml stock solution
10x Gel loading dye
DNA marker solution, DNA sample and gloves.
PROCEDURE:
1. Making a 1% Agarose Gel ( it will reach th lab as powder )
Weigh 0.5 g agarose and dissolve it in 50 mL of 1x TBE Buffer (running buffer).
(Note: Use 250 ml conical flask for preparing 50 ml solution to avoid
overflow of gel solution while heating and to avoid its loss.)
Heat the solution over a hot plate to boiling constituency marked
with a clear solution
Potent mutagen
Leave the solution to cool and add 2l of EtBr solution mix it well by Intercalating
agent
gentle swirling.
Go between the
Pour it in the gel tray-comb set up ( caster ). Also be sure the gel
DNA
plates have been taped securely and contain the well combs prior to
pouring ( in record : put it in casters then put the comb. I will make sure which to do
first from the doctor ) .
Allow the solution to cool and harden to form gel.
Make sure when you pour the solution that there is no air bubbles, because these
bubbles will affect the DNA movement in the gel.

2. Loading of Samples
Carefully transfer the gel to the electrophoresis tank filled with 1x 1xTBE buffer is the
running buffer here
TBE buffer ( electrophoresis chamber ).
Prepare your samples [9 ul of DNA sample (0. 1 ug to 1 ug) and 1 ul
of 5x gel loading dye]
Remove the comb and load the samples into the well.
Connect appropriate electrodes to the power pack and run it at 50100volts for 20min.
Monitor the progress of the gel with reference to tracking dye (Bromophenol
blue). Stop the run when the marker has run 3/4 th of the gel.
The DNA will move to the anode because It is an anion
3. Examining the gel
Place the gel on the UV-transilluminator and check for orange colored bands in
the gel.
Part two: Restriction Enzyme Digestion
In 1978, Daniel Nathans, Hamilton Smith, and Werner Albert received the Nobel Prize
in Physiology and Medicine for their work on enzymes that cut DNA at specific
sequences of nucleotides. These enzymes, known as restriction endonucleases, are
critical to any studies involving gene cloning and analyses as they allow you to cut up
larger pieces of DNA into smaller fragments with known ends. called "restriction
enzymes because restrict host range for certain bacteriophage. Used by the
bacteria as part of bacterial immune system to destroy and degrading any foreign
DNA. Bacteria protect their own DNA from being degraded by covalently attaching a
methyl group to adenines or cytosines within the recognition sequence
(restriction/modification systems).
Hundreds of restriction enzymes have been identified and isolated from prokaryotes.
Often, restriction enzymes recognize and cut palindromic sequences (sequence of
one DNA strand is identical to that of the complementary strand) and leave blunt
ends or staggered (sticky) ends. The Importance of the restriction enzymes relay on
their ability to create unpaired "sticky ends" which it can anneal with any
complementary sequence thus can used in downstream application such as RFLP
analysis (Restriction Fragment Length Polymorphism), DNA sequencing, DNA
storage libraries, Transformation, and restriction maps.
When we do DNA cloning or recombination we must use the same restriction
enzyme.

Named for bacterial genus, species,

strain, and type.
Example: EcoR1
Genus:Escherichia
Species:coli
Strain:R
Order discovered: 1
Summary of factors affecting the movement of DNA in gel
The factor
Size
Concentration of gel
Voltage
Conformation

Direct or inverse Description

proportion
Inverse
The smaller te faster
Inverse
More concentration the slower the
separation
Direct
The higher voltage the faster the
movement
Liner is faster
Nicked or open circular DNA will move
slowler than linear and super coiled DNA.

Our lab working

We will start loading samples in wells
1- Lambda DNA
2- Genomic DNA
Then we cut them with restriction enzymes, and we expect to see more fragments
in human genomic DNA, why ??? because it is larger so the probability to have
restriction site is much higher more fragment appear like smear ( ) .
3- we will load lambda DNA which had been cut with BamH I
4- we will load lambda DNA which had been cut with HinD III
5- we will load lambda DNA which had been cut with both BamH I & HinD III .
We expect to see more fragments with both enzymes
6 Human genomic DNA with both restriction enzymes

Here, we didnt add any RE

so we get 1 fragment and
it's very large fragment so it
didn't move.

We already load Ethidium Bromide(EtBr) in the gel, this dye is sensitive for uv-light.
Therefore we can use uv-light to visualize the DNA fragments. ( in research labs they
use X-ray )

After separation it will look like this:

Done By: Hothaifa AL-Jarrah