What is agarose gel electrophoresis. Its source, history, principle.
Equipment needed for Gel electrophoresis, and protocol.
Applications and pros and cons of Agarose gel electrophoresis
3. INTRODUCTION
Agarose gel electrophoresis is an easy and
efficient method to separate, identify, and
purify the DNA molecules
.
Agarose Gel Electrophoresis is a technique
used to separate DNA and RNA.
The material being separated is placed into a
gel-like substance called Agarose.
This is achieved by moving negatively
charged molecule through an Agarose matrix
with an electric field.
Shorter molecules faster than the longer ones.
4. HISTORY OF ELECTROPHRESIS
• In 1975 J.kalose and Farrell developed the 2D
Electrophoresis
• In 1981 J.W Jorgensen and K.D performed
electrophoresis for the separation of amino acids at the
high efficiency
• In 1990 Karger’s group discovered a matrix that could
be used to separate DNA at high resolution
• All these improvements led to the development of use
of electrophoresis in the mapping of human genome.
• 2000 to Now; Gel electrophoresis is the widely used
high resolution technique for analytical and preparative
solutions.
5. WHAT IS AGAROSE?
Meaning of Agarose is a ‘polysaccharide’
obtained from agar and used as a
supporting medium in gel electrophoresis.
A polysaccharide that can be used to form a
gel to separate molecules based on the size.
Agarose is a polysaccharide, generally
extracted from certain red seaweed.
It is a linear polymer made up of the
repeating unit of agarobiose.
Agarose is frequently used in molecular
biology for separation of large molecules.
6. PRINCIPLE
Powerful separation method used to analyze
DNA fragments generated by restriction
enzymes
Negatively charged DNA molecules migrates
towards the positive, under the influence of
electric current
The separation of DNA molecules depends on
Shape
Charge
Size
7. ELECTROPHRESIS EQUIPMENT
• Gel casting tray
• Gel tank
• Power supply
• Buffer
• Staining agent (dye)
• Comb
• DNA ladder
• Sample to be separate
• Electrophoresis chamber
8. GEL PREPRATION
Weigh out the appropriate mass of Agarose
into an flask.
Dissolve Agarose powder in
electrophoresis buffer.
Boil to dissolve the Agarose
Pour into gel trays and insert comb to form
loading wells.
Allow to harden at room temperature.
9. METHOD FOR ELECTROPHRESIS
Prepare the Agarose gel
Melt, cool and add Ethidium bromide, Mix thoroughly
Pour into casting tray with comb and allow it for solidify
Add running buffer, load sample and marker
Run gel at constant voltage until band separation occurs
Visualize DNA on UV illuminator and observe results
11. RESULTS
After separation, the resulting DNA
fragments are visible as clearly defined
bands.
The DNA standard or ladder should be
separated to a degree that allows for the
useful determination of the sizes of sample
bands.
Smaller pieces of DNA travel further than
larger pieces of DNA.
12. FACTORS AFFECTING THE GEL
ELECTROPHRESIS RESULTS
• Composition and concentration of buffer
• Concentration of Agarose gel
• Purity and concentration of DNA
• Voltage of electrophoresis
• Use of the buffer and Agarose gel
Electrophoresis
• Preparation of Gel
• pH of buffer and DNA
13. PROS/ CONS OF ELECTROPHRESIS
• High separation efficiency
• Short analysis time
• Low sample and electrolyte consumption
• DNA do not get ruined in the process
• Ease of operation
• Expensive
• Uses hazardous material
• Time consuming
14. GEL ELECTROPHRESIS IS USED TO
Solve criminal cases
Solve paternity cases
Diagnose genetic disease
Determine genetic kinship among species
15. CONCLUSION
Gel electrophoresis allows
us to visualize the sizes of
DNA segments and aids in
the sequencing of lengths
of DNA.
In gel electrophoresis,
negatively charged DNA
fragments travel through
the porous gel towards the
positive end of the tray
when a charge is applied
to the gel
