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The agarose concentration is varied for different fragment ranges. For analysing the complete β2-adrenoceptor coding region (1239 bp), a 1% agarose gel9 is made by dissolving agarose in 1Õ TAE buffer by heating in a microwave oven. After cooling to about 60℃C, ethidium bromide is added to a final concentration of 0.5 µg/ml. The agarose solution is poured into a taped gel former mold to make the gel. A well-forming comb (12 or 24 slots for minigels) is placed near one edge of the gel. The gel is cooled to harden until it becomes milky and opaque (approximately one hour). The gel mold is placed horizontally into the electrophoresis tank, which is filled with 1Õ TAE 10 (0.5 µg/ml ethidium bromide).
The gel loading buffer is applied to the samples and they are carefully added to individual wells11. The electrophoresis is run by 70–100 V/20–80 mA for about an hour or at 20 to 30 V overnight. The size of fragments can be determined by calibrating the gel, using known standards (e.g., λ DNA EcoRI / HindIII digest, Boehringer Mannheim, or 100bp ladder, BioVentures, Inc.), and comparing the distance the unknown fragment has migrated.
The most convenient method of visualising DNA in agarose gels is by use of the fluorescent dye ethidium bromide [118] (2,7-Diamino-10-ethyl-9-phenyl-phenanthridinium bromide). This substance (Figure 2.1) contains a planar group that intercalates between stacked bases of DNA. The fixed position of this group and its close proximity to the bases causes dye bound to DNA to display an increased fluorescent yield compared to dye in free solution. UV-irradiation absorbed by the DNA at 260 nm and transmitted to the dye, or irradiation absorbed at 300 nm and 360 nm by the bound dye itself, is emitted at 590 nm in the red-orange region of the visible spectrum.
Ethidium bromide can be used to detect both single- and double-stranded nucleic acids. However, the affinity of the dye for single-stranded nucleic acid is relatively low and the fluorescent yield is poor.
© 2001 Alexander Binder
