Agarose Gel Electrophoresis
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Transcript of Agarose Gel Electrophoresis
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Agarose Gel Electrophoresis
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Purposes To understand the principle of Gel
electrophoresis To become familiar with the part of the
electrophoresis setup
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Electrophoresis is a laboratory technique for
separating molecules based on their charge.
What is Electrophoresis?
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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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How Separation Occurs1- Electrical Charge:
Many molecules (amino acids, proteins, DNA, and RNA) have naturally occurring negative and positive charges on them.
The sum of these charges determines the overall charge.
Molecules with a negative charge (anions) will be attracted
to the positively charged node (anode).Molecules with a positive charge (cations) will be attracted
to the negatively charged node (cathode).
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2- 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.
Porous Material
Proteins Entering Porous Material
Smallest Move Fastest
How Separation Occurs
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• Agarose – a complex sugar chain from red seaweed.
• It is commonly used in foods (ice cream, and jellies) and many biological mediums.
• It has a large pore size good for separating large molecules quickly.
• Polyacrylamide – chain of acrylamide molecules.
• It is often used to make plastics and rubber.
• It has a small pore size good for separating small molecules. Acrylic Acid
Gels can be made from substances such as agarose or polyacrylamide.
Red Sea Weed
Gel Electrophoresis
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Agarose Gel A porous material derived from red seaweed Agarose is highly purified to remove
impurities and charge Acts as a sieve for separating molecules. This solid matrix will allow the separation of
fragments by size.
Concentration affects molecules migration Low conc. = larger pores better
resolution of larger DNA fragments High conc. = smaller pores better
resolution of smaller DNA fragments
1% agarose
2% agarose
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Fragment Resolution
% Agarose DNA fragment, kb
0.5 30-1 0.7 12-0.8 1.0 10-0.5 1.2 7-0.4 1.5 3-0.2
Gel Concentration – Is dependant upon the size of the DNA fragments to be separated.
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+-
Power
smalllarge
• Agarose at Room Temperature is a 3-Dimentional solid matrix.
• The smaller the fragments the further the migration or movement through the matrix.
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Purposes for Agarose Gel Electrophoresis
• Analysis of molecules size • Separation and extraction of molecules• Quantification of molecules
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Procedure
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Components of an Electrophoresis System
Power supply and chamber, a source of power supply
Buffer, a fluid mixture of water and ions
Agarose gel, a porous material that molecules migrates through
Gel casting materials
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BufferDyes Power Supply
+
-Cathode
Anode
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Electrophoresis Buffer
TAE (Tris -acetate-EDTA) and TBE (Tris-borate-EDTA) – pH buffer
Tris Acetic acid provide ions to support conductivity and maintain pH
EDTA, prevent brake down of molecules
Concentration affects DNA migration Use of water will produce no migraton High buffer conc. could melt the agarose gel
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• Gel Preparation
• Loading the gel
• Running the gel
Overview of Agarose Gel Electrophoresis
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Agarose is a linear polymer extracted from seaweed.
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Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.
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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.
Melting the Agarose
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Migration of molecules in Agarose
Rate of migration of a molecule is inversely proportional to the log of its molecular weight
Distance α 1 / log-MW
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Distance (mm)
Log-
Mol
ecul
ar W
eigh
t
1
2
3
Best Fit Line