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Unformatted text preview: Laboratory: X Topic : Agarose Gel Electrophoresis of DNA Introduction : The technique is based on the fact that molecules (DNA and RNA) possess a net negative charge and can therefore migrate when placed in an electric field. The porous agarose gel is the matrix through which the DNA or RNA molecules when placed in buffer solution. Electrodes are attached to each end of the buffer chamber, one becoming the positively charged anode , and the other becoming the negatively charged cathode . Any positively charged particle (cation) will migrate toward the cathode, and any negatively charged particle (anion) will migrate toward the anode. Depending on the size of the fragments the distance of migration through the gel varies. Small fragments migrate quickly and therefore are observed further from the wells. For better resolution of larger fragments the running time is increased. The concentration of the agarose can also be adjusted depending on the size of the fragments Isolated plasmid DNA occurs as supercoiled, nicked or linear depending on the quality of the DNA prep and the restriction digestion. Uncut circular plasmid (undigested) is generally supercoiled and runs faster than linear DNA of the same size. In a poor quality DNA prep nicked as well as linear DNA is observed in the uncut prep. For same size DNA migration is as follows: well as linear DNA is observed in the uncut prep....
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- Spring '08