Gel Electrophoresis.

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Presentation transcript:

Gel Electrophoresis

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.

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.

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

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.

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

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).

Gel Electrophoresis Loading dye Visualization Glycerol Dye (bromophenol blue ~ 200 -400 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

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

Quality and Quantity Assessment

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.