Agarose Gel Electrophoresis

Purposes To understand the principle of Gel electrophoresis To become familiar with the part of the electrophoresis setup

What is Electrophoresis? Electrophoresis is a laboratory technique for separating molecules based on their charge and size .

Separation of a Mixture of Charged Molecules Charged molecules are separated based on their electrical charge and size. Charge Separation Size Separation Analyze Identify Purify Mixture of Charged Molecules Positive Molecules Negative Molecules

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).

Proteins Entering Porous Material How Separation Occurs 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. Size to molecular ratio Porous Material Proteins Entering Porous Material Smallest Move Fastest

Gel Electrophoresis Gels can be made from substances such as agarose or polyacrylamide. 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. Red Sea Weed Acrylic Acid

Agarose Gel 1% agarose 2% agarose 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

Fragment Resolution Gel Concentration – Is dependant upon the size of the DNA fragments to be separated.

+ - Agarose at Room Temperature is a 3-Dimentional solid matrix. The smaller the fragments the further the migration or movement through the matrix. + - Power small large

Purposes for Agarose Gel Electrophoresis Analysis of molecules size Separation and extraction of molecules Quantification of molecules

Procedure

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 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

- + Cathode Anode Buffer Power Supply Dyes 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). Anode + Buffer Power Supply Dyes

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 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.

Overview of Agarose Gel Electrophoresis Gel Preparation Loading the gel Running the gel

Agarose is a linear polymer extracted from seaweed.

Agarose Buffer Solution Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.

Melting the Agarose 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.

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

3 Best Fit Line 2 Log- Molecular Weight 1 Distance (mm)