this section helps students how to made agrose gel and how to run DNA molcules on agarose gel . specially life life science fields such as biotechnology, biology, and medical laboratory

1. University of Gondar
Institute of Biotechnology
Techniques in Biotechnology (Biot.602)
Lecture 7
Agarose Gel Electrophoresis

2. Definition
What is Gel Electrophoresis ???
• Mixture: a material composed of
two or more elements or parts.
• Charged Molecules: a molecule
(such as a protein or DNA) that has
too many or too few electrons.
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• Is a procedure that separating mixtures of
charged molecules based on their size due to
the influence of electric current.
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3. Charged molecules are separated based on their electrical charge and size.
Separation of a Mixture of Charged Molecules
Charge
Separation
Size
Separation
Analyze
Identify
PurifyMixture of
Charged
Molecules
Positive
Molecules
Negative
Molecules
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4. - - Negative Electrode -
-
+ + Positive Electrode + +
- - Negative Electrode - -
+ + Positive Electrode + +
Before Electrophoresis After Electrophoresis
Wells
AGE…
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5. AGE…
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These charged molecules may be:
DNA
RNA
Protein
These molecules will be separated based on
their migration rate.
The resistance to movement is due to the
sieving effect of the gel matrix
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6. AGE…
What is the purpose of using electrophoresis?
To Separate visualize:
– Genomic and plasmid DNA
– RNA
– PCR products
– Restriction enzyme digest products
– Protein
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• Agarose
 The Agarose for Agarose gel electrophoresis is
purified from agar.
 Agarose is a linear polymer made up of repeating
units of 1,3 –linked ß D galactopyranose and 1, 4
linked 3,6 anhydro L galactopyranose.
 The cross links are held together by hydrogen and
hydrophobic bonds.
 It has a large pore size good for separating large
molecules quickly.
What is Agarose?
Gels can be made from substances such as agarose or polyacrylamide.
Red Sea
Weed
AGE…
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8. Tadele T. 8
Casting tray
Gel combs
Power supply
Gel tank
Cover
Electrical leads

ElectrAGE… ophoresis Equipment
Electrophoresis Equipment
Combs'
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9. AGE…
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Seal the edges of the casting tray and put in the combs. Place the casting
tray on a level surface. None of the gel combs should be touching the
surface of the casting tray.
Gel casting tray & combs 1. Preparing the Casting
Tray
Making an Agarose Gel
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10. AGE…
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Agarose
Buffer Solution
An agarose gel is prepared by combining agarose powder and a buffer
solution. Either TBE or TAE.TBE is made with Tris/Boric Acid/EDTA.
TAE is made with Tris/Acetic Acid/ EDTA. A buffer is a substance that
resists changes in pH.
Agarose
Buffer
Flask for
boiling
2. Combining agarose powder and a buffer
solution
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11. AGE…
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Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
Gently swirl the solution periodically when heating to allow all the grains of agarose
to dissolve.
***Be careful when boiling - the agarose solution may become superheated and
may boil violently if it has been heated too long in a microwave oven.
3. Melting the Agarose
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12. AGE…
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• Allow the agarose solution to
cool slightly (~60ºC) and then
carefully pour the melted
agarose solution into the
casting tray. Avoid air bubbles.
Pouring the gel
• Each of the gel combs should
be submerged in the melted
agarose solution.
4. Pour the melted agarose solution into
the casting tray.
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13. AGE…
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When cooled, the agarose
polymerizes, forming a
flexible gel. It should appear
lighter in color when
completely cooled (30-45
minutes). Carefully remove
the combs and tape.
A series of wells used to
load the DNA.
6. Place the gel in the
electrophoresis chamber.
buffer 
7. Add enough electrophoresis buffer to cover the
gel to a depth of at least 1 mm. Make sure each
well is filled with buffer.
Cathod
e
(negative)
Anode
(positive)

wells
  
DNA
5. Removing the comb from the
hardened gel
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14. AGE…
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• Sample : Loading Buffer (5:1ul)
• 6X Loading Buffer:  3ul
 Bromophenol Blue (for color)
 Glycerol (for weight)
• Sample 12ul
 Total 15ul will be loaded in to the gel
8. Sample Preparation and Loading in to the Gel
• Preparation of sample loading dye Glycerol &
bromophenol blue (6x)
 3ml glycerol (30%), 25mg bromophenol blue
(0.25%) dH2O to 10mL
• Mix the samples of DNA with the sample loading buffer
 Bromophenol blue allows the samples to be seen
when loading onto the gel, as a front page
indicators and
 Glycerol increases the density of the samples,
causing them to sink into the gel wells.
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15. AGE…
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Loading the Gel
• Carefully place the pipette tip over a well and gently expel the
sample. The sample should sink into the well. Be careful not to
puncture the gel with the pipette tip.
The sample or DNA is loaded with a loading
buffer-containing dyes and glycerol or sugar
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• Place the cover on the electrophoresis chamber, connecting the
electrical leads to the power supply.
• Be sure the leads are attached correctly - DNA is negatively charged
(due to PO4). Migrates from the negative (black) electrode to the
positive (red) electrode.
• When the power is turned on, bubbles should form on the electrodes
in the electrophoresis chamber.
9. Running the Gel
Why Run a Gel?
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17. AGE…
Why Run a Gel?
• Comparison of sample bands to markers allow:
– Visible confirmation of desired product
– Quality determination of sample DNA
NB: Molecular weight markers are DNA
fragments of known size
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18. AGE…
• During electrophoresis water undergoes hydrolysis : H2O 
H+ and OH-
• The anode (+ /red) pole becomes alkaline because OH- will
accumulate at this pole
• The cathode (-/black) pole becomes acidic because H+ will
accumulate at this pole
• Buffers prevent the pH from changing by reacting with the H+
or OH- products
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19. AGE…
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10. Staining the Gel
• To make DNA fragments visible after electrophoresis, the DNA
must be stained.
• Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
– Which is quite sensitive to detects 0.1ug of DNA concentration.
• Ethidium bromide can be added
– To the gel and/or
– Running buffer before the gel is run or
– The gel can be stained after it has run.
 By convention it is besting adding of EtBr in the gel before pouring.
• Two big problems
UV light can damage your eyes----and need to be
wear safety googles!!!!!
Ethidium bromide is a mutagen!!!!!
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EtBr staining gel
• The standard concentration
used in staining DNA in gels
is 0.5-1ug/mL
• Sensitive—detects 0.1ug of
DNA
• Inexpensive--$0.02 per gel
• Stains in 10 minutes/or
immediate if in gel,
• Powder is especially
dangerous due to possible
inhalation and so premade
solutions are always
purchased
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21. AGE…
Alternatives to EtBr
Methylene Blue
• Sensitivity –Better than
0.5ug DNA
• Stain time 30 minute
• Can’t be added to gel
• Stain solution can be
reused
• Cost $0.20/gel
• Used white light
SYBER Safe
• Sensitivity –Better than
0.1ug DNA
• Stain time 30 minute
• Can’t be added to gel
• Stain solution can’t be
reused
• Cost $0.50/gel
• Used UV light
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22. AGE…
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A gel stained with
Methylene blue
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23. AGE…
• Rate of migration of DNA through agarose depends:
The Molecule Size:
– The porous material is made of microscopic particles
suspended in a gel. The microscopic particles attach to one
another forming tunnels that act as a sieve to separate the
molecules. Small molecules can move faster than large
molecules.
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Porous Material Proteins Entering
Porous Material
Smallest Move
Fastest
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24. Agarose concentrations
– Higher concentration of gels are used for the
lower molecular weight DNA and RNA paration of
fragments and vice-versa
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25. Conformation
• Supercoiled DNA moves fastest followed by
linear forms and relaxed open circular forms.
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26. AGE…
• The voltage applied to the gel
– Affects how quickly the gel runs
– The higher the voltage, the more quickly the gel runs………
But that often reduces the quality of the DNA
separation
It also generates heat which reduces the quality of the
DNA separation
The best separation will apply voltage at no
more than 5V/cm of gel length.
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27. Tadele T. 27
11. Visualizing the DNA
 100
 200
 300
 1,600
 1,000
 500
 850a
 600
 400
 5,000 bp
 2,000
DNA ladder

DNA ladder

PCR Product
1 2 3 4 5 6 7 8
wells
+ - - + - + + -
Samples # 1, 4, 6 & 7 were positive for amplification of DNA
Primer dimers
AGE…
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28. Summary of agarose gel electrophoresis
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Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix thoroughly.
Add running buffer, load samples and marker
Run gel at constant voltage until band separation occurs
Pour into casting tray with comb and allow to solidify
View DNA on UV light box and document results
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29. Thank you
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