Shefigem2010:Electrophoresis

Agarose gel electrophoresis of DNA:

This method allows for the separation of DNA molecules depending on their size. The phosphate backbone of DNA bears a negative charge and so the molecule will travel toward a positively charged anode. Ethidium bromide intercalates with DNA molecules, and is used as a tag, to visualise the bands, once the gel has run. A 1% agarose gel provides decent resolution for DNA fragments ranging in size from 0.2Kb – 10Kb. In order to make a 1% mini (10 x 8cm) agarose gel, 0.5g agarose was mixed with 50ml 0.5 x TBE. The agarose was dissolved by heating in an 850W microwave for 1-2 min. Once the mix had cooled down to ~50°C, 0.5ul (10mg/ml) ethidium bromide was added and the mixture was poured into a sealed, gel cast. The gel cast held a comb with the appropriate number of wells. The gel was allowed ~20 min to set at room temperature before it was placed into the Mini-Sub Cell GT Agarose Electrophoresis system [Bio-Rad, Bio-rad Laboratories., Heidenmannstrase, Munchen, Germany], containing 0.5 x TBE. DNA samples were prepared by mixing with 6 x loading buffer to the working concentration (1 x). The samples were loaded along with a commercial 1Kb DNA marker [GeneRulerTM, Fermentas UK, York]. The gel was run at a constant 100 volts until the dye front reached the bottom of the gel. Ethidium bromide fluoresces under UV light, so bands were visualised under exposure to this wavelength.