Biology SPA
Agarose gel Electrophoresis
Used to separate and purify macromolecules
 e.g. proteins and nucleic acids that differ in size, charge of conformation
When charged molecules are placed in an electric field, they migrate towards either the positive (anode) or
negative (cathode) pole.
Proteins – either a net positive or net negative charge
Nucleic acids – Consistent negative charge imparted by their phosphate backbone (migrate towards anode)
Electrophoresis buffer which gel is immersed in:
-
Provides ions to carry a current
Maintain the pH
Fragments of DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their
molecular weight. Smaller = faster, larger = slower
Uncut plasmids will migrate more rapidly than the same
plasmid when linearized.
Uncut plasmid contains at least 2 topologically different
forms of DNA, supercoiled forms and nicked circles
.
Higher concentrations of agarose facilitate the
separation of small DNAs, while low agarose
concentrations allow resolution of larger DNAs.
As the voltage applied to a gel is increased, larger fragments migrate proportionally faster than smaller fragments.
DNA staining with Ethidium Bromide (dye)
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DNA can be detected as visible fluorescence when gel is illuminated with ultraviolet light
DNA absorbed UV light very efficiently
Ultraviolet Spectrophotometry of DNA
Nucleotides – Maximum absorption of 260 nm
Proteins – Maximum absorption of 280 nm
Absorbance of a DNA sample at 280 nm gives an estimate of the protein contamination of the sample
The ratio of A260: A280 is a measure of the purity of a DNA sample. It should be between 1.65 and 1.85.