Agarose Gel Electrophoresis

1. Agarose Gel
Electrophoresis

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

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

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

12. Procedure

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

14. Buffer
Dyes Power Supply
+
-
Cathode
Anode

15. Casting tray
Gel combs
Power supply
Gel tank
Cover
Electrical leads

Electrophoresis Equipments

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

21. Gel casting tray & combs

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.

25. Place the gel in the electrophoresis chamber.

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.

28. Running the Gel

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

30. Distance (mm)
Log-MolecularWeight
1
2
3
Best Fit Line