Agarose Gel Electrophoresis

Purposes To understand the principle of Gel electrophoresis. To become familiar with the part of the electrophoresis setup. Electrophoresis is a laboratory technique for separating molecules based on their charge and or size. This technique is simple: Biological samples are prepared and placed in gel (matrix). Electricity is then run through the matrix, causing molecules in the samples to separate.

There are two components of electrophoresis system: Electrical filed. The physical resistance of the matrix. Electrical filed 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. At neutral pH: proteins have a unique electrical charged. both DNA and RNA bases are negative charged. Molecules with a negative charge (anions) will be attracted to the positively charged node (anode)……. Red color. Molecules with a positive charge (cations) will be attracted to the negatively charged node (cathode)…… Black color.

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

The physical resistance of the matrix Electrophoresis is conducted through a gel substances called a matrix. Matrix acts as a physical barrier to movement of the substances. This barrier acts as a ”sieve” to separate the molecules by size. Without any resistance to there movement, “equally charged molecules will migrate through the electrical field at the same rate. If we put barrier in there way small molecules can move more quickly than large one.

Proteins Entering Porous Material Molecule Size: The molecule 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 is made of microscopic particles Porous Material Proteins Entering Porous Material Smallest Move Fastest

Components of an Electrophoresis System Power supply and chamber: a source of power supply Agarose gel: a porous material that molecules migrates through Buffer: a fluid mixture of water and ions. 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 Power Supply

- + Cathode Anode Buffer 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 Dyes

Gel Electrophoresis They are many types of matrices used in electrophoresis, but all function similarly as physical barrier to movement. Gels can be made from substances such as agarose or polyacrylamide. Agarose “ a complex sugar chain from red seaweed”. Non toxic carbohydrate. 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.

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

Fragment Resolution Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1% gels are common for many applications. DNA fragment, kb % Agarose 30-1 0.5 12-0.8 0.7 10-0.5 1.0 7-0.4 1.2 3-0.2 1.5

+ - 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

Electrophoresis Buffer TAE (Tris -acetate-EDTA) and TBE (Tris-borate-EDTA) – pH buffer. Tris a pH buffer. Acetic acid provide ions to support conductivity and maintain pH. EDTA, prevent brake down of molecules. “all dissolved in water”. The important feature of any buffer is that it contains an electrolyte so that it can conduct electricity. 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.

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

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

Gel Preparation 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.

Loading the gel

Running the gel