KMU Gel Eletrophrosis

AGAROSE GEL ELECTROPHORESIS
MUHAMMAD TARIQ
Medical Technologist, Dept of Blood Bank (SBT)
HMC Peshawar
M Sc. (Medical Technology). PhD. Fellow
(Genetics)
ELECTROPHORESIS
• Electro means flow of electricity while phoresis is

from Greek word which means to carry across.
• Electrophoresis is a term used to describe the

migration of a charged particle under the
influence of an electric field through solution.
AGAROSE GEL ELECTROPHORESIS
• A method used in biochemistry and molecular
biology to separate DNA or RNA molecules by size.
This is achieved by moving negatively charged
nucleic acid molecules through an agarose matrix
with an electric field (electrophoresis). Shorter
molecules move faster and migrate farther than
longer.
PRINCIPLES OF GEL ELECTROPHORESIS
• Electrophoresis is a technique used to separate and
sometime purify macromolecules.
 Protein and nucleic acids that differ in size, charge or
conformation.
• Charged molecules placed in an electric field migrate
toward either the positive (anode) or negative
(cathode) pole according to their charge.
• Protein and nucleic acid are electrophoresed within a
matrix or “gel”.
Why do we
use gel electrophoresis?
• Gel electrophoresis is an important tool in biology.
• It can be used to identify specific DNA molecules that

have been isolated and cut up by restriction
enzymes.
• We also use it to determine differences in the

genomes of different plant and animal species.
Cont…
• Gel electrophoresis is also used in forensic science to
compare the DNA fingerprints found at crime scene
and that of the suspect.
• DNA samples collected from blood or semen are

separated using gel electrophoresis.
• The number and positions of the bands formed on

each lane of gel is the actual DNA fingerprint and is
unique to each person.
Equipments and supplies
• Buffer solution, usually TBE buffer or TAE 1.0x. pH 8.0
• Agarose.
• An ultraviolet-fluorescent dye, ethidium bromide, (5.25
mg/ml in H2O). Alternative dyes may be used, such as SYBR
green.
• Nitrile rubber gloves. Latex gloves do not protect well from
ethidium bromide.
• A color marker dye containing a low molecular weight dye
such as “ bromophenol blue ”.
• A gel rack.
• A “comb”.
• Power supply.
• UV lamp OR UV lightbox or other method to visualize DNA in
the gel.
Making an Agarose Gel
• An agarose gel is
prepared by combining
agarose powder and a Buffer
buffer solution.
Agarose
Flask for boiling

Electrophoresis Equipment
Power supply
Cover
Gel tank
Electrical leads

Gel combs
Gel casting tray & combs
Preparing the Casting Tray
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.
Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.
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.
Pouring the gel
Allow the agarose solution to cool slightly (~60ºC) and then carefully
pour the melted agarose solution into the casting tray. Avoid air
bubbles.
Each of the gel combs should be submerged in the melted agarose solution.
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.
Place the gel in the electrophoresis chamber.
DNA
buffer     
wells
Anode
Cathode (positive)
(negative)
Add enough electrophoresis buffer to cover the gel to a depth of

at least 1 mm. Make sure each well is filled with buffer.
Sample Preparation
Mix the samples of DNA with the 6X sample loading buffer (w/ tracking
dye). This allows the samples to be seen when loading onto the gel,
and increases the density of the samples, causing them to sink into the
gel wells.
6X Loading Buffer: 
 Bromophenol Blue (for color)
 Glycerol (for weight)
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.
Running the Gel
Place the cover on the electrophoresis chamber, connecting the electrical

leads. Connect the electrical leads to the power supply. Be sure the leads
are attached correctly - DNA migrates toward the anode (red). When the
power is turned on, bubbles should form on the electrodes in the
electrophoresis chamber.
Cathode
(-)
 wells
DNA  Bromophenol Blue
(-)

Gel
Anode
(+)
After the current is applied, make sure the Gel is running in the correct
direction. Bromophenol blue will run in the same direction as the DNA.
DNA Ladder Standard
-
 12,000 bp
 5,000
DNA
migration  2,000
 1,650
Note: bromophenol
blue migrates at  1,000
approximately the  850
same rate as a 300 bp  650
DNA molecule  500
 400
bromophenol blue  300
 200
+  100
Inclusion of a DNA ladder (DNAs of know sizes) on the gel

makes it easy to determine the sizes of unknown DNAs.
FACTORS AFFECTING MIGRATION
1. DNA or RNA molecular weight.
2. Voltage.
3. Agarose.
4. Buffer.
5. Visualisation.
Staining the Gel
• Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
• 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.
***CAUTION! Ethidium bromide is a powerful mutagen and is

moderately toxic. Gloves should be worn at all times.
Safer alternatives to Ethidium Bromide
 Methylene Blue
 BioRAD - Bio-Safe DNA Stain
 Ward’s - QUIKView DNA Stain
 Carolina BLU Stain
…others
advantages disadvantages
Inexpensive Less sensitive
Less toxic More DNA needed on gel
No UV light required Longer staining/destaining time
No hazardous waste disposal
Staining the Gel
• Place the gel in the staining tray containing warm diluted stain.
• Allow the gel to stain for 25-30 minutes.
• To remove excess stain, allow the gel to destain in water.
• Replace water several times for efficient destain.
Ethidium Bromide requires an ultraviolet light source to visualize
Visualizing the DNA (ethidium bromide)
DNA ladder DNA ladder
 1 2 3 4 5 6 7 8 
wells
 5,000 bp
 2,000
 1,650
 1,000
 850
 650
 500
PCR Product  400
 300
 200
Primer dimers  100
+ - - + - + + -
Samples # 1, 4, 6 & 7 were positive for Wolbachia DNA
Visualizing the DNA (QuikVIEW stain)
DNA ladder

wells
PCR  2,000 bp
 1,500
Product  1,000
 750
 500
 250
+ - - - - + + - - + - +
Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia DNA

March 12, 2006
ADVANTAGES AND DISADVANTAGES
1. ADVANTAGES
• The gel is easily poured.
• It does not denature the samples.
• The samples can also be recovered.
2. DISADVANTAGES
• Gel can melt during electrophoresis.
• The buffer can become exhausted and different
forms of genetic material may run in un predictable
forms.
Q1.We use electrophoresis to:
a) Identify DNA fragments cut by restriction enzymes
b) Determine differences in in the genome of different plants and animals species
c) Compare the DNA finger prints found at crime scene and that of the suspect
d) Detect different DNA Mutation
e) All of the above
Q2.Equepments use in electrophoresis are all except:
a) TBE Buffer
b) Power Supply
c) Gel Tank
d) Gel Casting tray
e) Gel Combs
Q3.While running the gel the DNA moves from:
a) Cathode to Anode
b) Anode to Cathode
c) Both of the above
d) None of the above
e) All of the above
Q4.Factors affecting migration of DNA/RNA are all except:
a) DNA or RNA molecular weight.
b) Voltage.
c) Agarose.
d) Buffer.
e) Gel Comb
Q5.Following are the safer alternative to Ethidium Bromide except:
a) Methylene Blue
b) BioRAD-Bio-Safe DNA Stain
c) Fluorescent Ethidium Bromide
d) Ward’s-QUIKView DNA Stain
e) Carolina BLU Stain
Thanks

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AGAROSE GEL ELECTROPHORESIS

• Electro means flow of electricity while phoresis is

• Electrophoresis is a term used to describe the

• It can be used to identify specific DNA molecules that

• We also use it to determine differences in the

• DNA samples collected from blood or semen are

• The number and positions of the bands formed on

Flask for boiling

Add enough electrophoresis buffer to cover the gel to a depth of

Place the cover on the electrophoresis chamber, connecting the electrical

Inclusion of a DNA ladder (DNAs of know sizes) on the gel

***CAUTION! Ethidium bromide is a powerful mutagen and is

Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia DNA

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