Transcript الشريحة 1
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. Separation of a Mixture of Charged Molecules Charged molecules are separated based on their electrical charge and size. Positive Molecules Analyze Charge Separation Size Separation Mixture of Charged Molecules Identify Purify 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). 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. 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. Red Sea Weed • 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 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 1% agarose 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 2% agarose Fragment Resolution Gel Concentration – Is dependant upon the size of the DNA fragments to be separated. % Agarose 0.5 0.7 1.0 1.2 1.5 DNA fragment, kb 30-1 12-0.8 10-0.5 7-0.4 3-0.2 • Agarose at Room Temperature is a 3-Dimentional solid matrix. • The smaller the fragments the further the migration or movement through the matrix. small large - Power + 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 Cathode Anode + Buffer Dyes Power Supply Electrophoresis Equipments Power supply Cover Gel tank Electrical leads Casting tray Gel combs 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 Overview of Agarose Gel Electrophoresis • Gel Preparation • Loading the gel • Running the gel Gel Preparation Agarose is a linear polymer extracted from seaweed. 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. Gel casting tray & combs 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. 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. Place the gel in the electrophoresis chamber. 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. Running the Gel 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 Log- Molecular Weight Best Fit Line 2 1 Distance (mm)