4. 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
5. How Separation Occurs
1- 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).
6. 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
7. • 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
8. 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
9. 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.
10. +-
Power
small
large
• Agarose at Room Temperature is a 3-Dimentional
solid matrix.
• The smaller the fragments the further the
migration or movement through the matrix.
11. Purposes for Agarose Gel Electrophoresis
• Analysis of molecules size
• Separation and extraction of molecules
• Quantification of molecules
13. 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
16. 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
17. • Gel Preparation
• Loading the gel
• Running the gel
Overview of Agarose Gel
Electrophoresis
18. Agarose is a linear polymer extracted from seaweed.
Agarose is a linear polymer extracted from seaweed.
Gel Preparation
19. Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.
20. 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
23. 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.
24. When cooled, the agarose polymerizes, forming a flexible gel. It should
appear lighter in color when completely cooled (30-45 minutes).
Carefully remove the comb.
26. 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.
29. 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
Gel concentration
Shape
Voltage
Interaction with agarose
Size to molecular ratio
Ions: atoms that have a positive or negative charge because they have lost or gained electrons.
Electrophoresis: migration of ions at different speeds is a basic principal
During electrophoresis, water is electrolyzed which generates protons (H+ ions)at the anode (positive) and hydroxyl ions (OH -1)at the cathode (negative). The cathode (negative) end of the electrophoresis chamber then becomes basic and the anode (positive) end becomes acidic.
The electrode at which electrons enter the gel box from the power supply (along the black wire) is called the cathode and is negative (-). The electrode at which electrons leave the box and re-enter the power supply (along the red wire) is called the anode and carries a positive charge (+). The flow of electrons sets up a potential energy difference between the electrodes. This is known as potential, and is measured in volts. It establishes an electric field through which the ions in the gel box fluid migrate. The migration of ions in the fluid creates electrical current which is measured in milliamperes (milliamps).
A buffer is a chemical system that maintains a relatively constant pH even when strong acids or bases are added. Buffer solutions contain either a weak acid or weak base and one of their salts. Because a change in pH can alter the charge on a particle, it is important to use a buffer solution when separating during electrophoresis.