1. Gel Electrophoresis

2. Gel Electrophoresis
Electrophoresis allows separation of molecules in an electrical field on the basis of size/molecular/weight and shape.
A molecule with a negative charge (anion) will migrate toward the positive electrode (anode), and a molecule with a positive charge (cation) will migrate toward the negative electrode (cathode)
The migration and separation of molecules are carried out using a solid matrix (i.e. agarose, polyacrylamide).
The matrix retards the movement of molecules by a seiving effect – small molecule navigate the matrix more quickly than larger ones.

3. Gel Electrophoresis
Electrophoresis is used chiefly for analysis and purification of large molecules such as proteins and nucleic acids, but can be a applied to any charged molecules.
Nucleic acids are separated using either polyacrylamide or agarose as the matrix, depending on the sizes of the molecules to be analyzed. For now, we will be separating DNA fragments using agarose as the matrix
The relative mobility of the fragments– how fast they travel though the matrix relative to each other - will depend on several parameters.

4. Gel Parameters Migration through the gel can be effected by: Size
Shape
% Agarose
Voltage

5. Gel Parameters Size Shape % Agarose
Smaller fragments of DNA will travel further than the larger ones.
Shape
Bacteria possess extrachromosomal circular DNA known as plasmids. Isolated plasmids come in three distinct shapes
% Agarose
Fragments larger than the matrix pore size cannot enter the gel and are not resolved. At the other extreme, fragments smaller than the pore size are not retarded at all.

6. Gel Parameters Voltage P=VI
In addition to Ohm’s law (V=IR), a fundamental equation in electrophoresis is the power equation
P=VI
The higher the voltage, the greater the power – heat!
gel melts
Non-uniform heat distribution results in smiling bands (heat is more rapidly dispersed at edges of gel)
5 – 8 cm /V

7. Gel Parameters Other Considerations Buffers and pH
During electrophoresis water is electrolyzed, generating protons (H+) at the anode, and hydroxyl ions (OH-) at the cathode. This results in regions of high and low pH. Therefore a buffer is needed.
The two most popular buffers for agarose gel electrophoresis are Tris-Borate-EDTA (TBE) and Tris-Acetate-EDTA (TAE).

8. Gel Electrophoresis Loading dye Visualization Glycerol
Dye (bromophenol blue ~ bp)
Visualization
Ethidium Bromide is a dye that intercalates between the base of nucleic acids (DNA and EtBr travel in opposite orientation). When it does, there is a shift in its absorption spectrum and it will fluoresce under UV light.
Dye

9. Quality and Quantity Assessment
Gel electrophoresis
Quantity Band intensity is semi-quantitative
Quality High molecular weight DNA (single band) Sheared DNA indicates poor quality DNA (smear)
Spectroscopy
A260 = 50 ug/ml
A260/280 > 1.5

10. Quality and Quantity Assessment

11. Markers
In agarose gel electrophoresis there are many parameters to consider
% agarose
Voltage
Molecular Conformation (Shape)
Size
Thus the molecules separate by size relative to the conditions in which the electrophoresis was conducted.
A DNA marker, a sample of DNA fragments of known sizes and mass, is used as a reference to estimate the size of unknown DNA molecules.