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.

What is Electrophoresis?

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

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).

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

• 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

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

% 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.

+-

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.

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

Buffer

Dyes Power Supply

+

-Cathode

Anode

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

• Gel Preparation

• Loading the gel

• Running the gel

Overview of Agarose Gel Electrophoresis

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.

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

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

Distance (mm)

Log

- M

ole

cula

r W

eig

ht

1

2

3

Best Fit Line