1. Dr. Israa ayoub alwan Lec -11-
AL-Ma’moon University College Medical Laberatory techniques Department Molecular biology/ Second stage
Dr. Israa ayoub alwan
Lec -11-

2. Gel electrophoresis

3. Gel electrophoresis: - is a technique used for separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix. It is usually performed for analytical purposes, but may be used as a preparative technique prior to use of other methods such as, RFLP, PCR, cloning, DNA sequencing.

4. The method is widely used to:-
1- Determine the molecular weight of DNA, RNA, and proteins
2- Determine the size of most DNA molecules and proteins
3- Determine the shape of DNA (bacterial DNA)
Circular closed molecules (cc)
Open circular (op)
Linear

5. The movements of charged molecules in buffer depend on:-
Size of molecules
Shape of molecules
charge of molecules
pore size present in the agarose gel
electrophoresis buffer

6. The term ''gel'' refers to matrix used to contain then separate the target molecules. In most cases, the gel is across linked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually composed of polyacrylamide. When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose

7. ''Electrophoresis'' refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, usually determined by mass, toward the positive anode if negatively charged or toward the negative cathode if positively charged.  

8. Agarose gel electrophoresis
Is a natural colloid extracted from seaweed composed of long linear chains of multiple saccarides (sugar).
It melts at high temperature in water and salts buffer solution.
It is very fragile and easily destroyed by handling.
Agarose gel has very large pore size and used primarily to separate very large molecular mass greater than 200kdal.
Agarose gel can be processed faster than polyacrylamide gels but their resolution is inferior the bands formed in agarose gel are fuzzy and separate far apart.
It is usually used at concentrations between 1%and 3%
Agarose gel is formed by suspending dry agarose in aqueous buffer then boiling the mixture until a clear solution is forms then poured and allowed to dry at room temperature.

9. Polyacrylamide gel electrophoresis (PAGE):- Is a technique introduced by Raymond Weintraub(1959) to provide a wide variety of electrophoreses condition.
It is more effective for separating small fragments of DNA (50-500bp).
Its resolving power is high and DNA fragments that as little as 1bp can be separated from one another.
The pore size for the gel may be varied to produce different molecular sizing for separating proteins of different sizes.
The percentage of Polyacrylamide can be controlled in a given gel by controlling the percentage (from 3%-30%) to obtain Precise pore size usually from 5 to 2000 kdal this is ideal range for gene-sequences, protein, polypeptide and enzymes analysis.
It offer greater flexibility and more sharply defined banding than agarose gels.
It is more difficult to prepare and handle than agarose.

10. Electrophoresis unit consist the following
1. Tank or cell
2. Tray
3.Comb
4-UV cabinet
The materials used
1. TBE buffer
2. Stock sol.
agarose
4. Loading buffer
5. Ethidium bromide stain

14. THANK YOU