Visualization on agarose gels

10 Apr

Visualization on agarose gels

A relatively quick and easy method for assessing both the quantity and quality of extracted DNA is to visualize it on an agarose gel. Agarose is a polymer that can be poured into a variety of gel forms – mini gels approximately 10 cm long are sufficient to visualize DNA. The gel is submerged in electrophoresis buffer and the DNA is loaded into wells that are formed in the gel by a comb; an electric current is applied across the gel and the negatively charged DNA migrates towards the anode. The agarose gel forms a porous matrix and smaller DNA molecules move through the gel more quickly than do larger DNA molecules. Dyes that intercalate with the DNA double helix, such as ethidium bromide [11], can be added to the gel either before or after electrophoresis, the amount of intercalated dye is proportional to the quantity of DNA. An alternative dye, DAPI (4′,6-diamidino-2-phenylindole), can be added directly to the DNA before electrophoresis. This migrates through the gel bound to the minor groove of double strandedDNA[37].DNAisvisualizedbyplacingthegelonatransilluminatorthatemits UVlightat260nm.Quantificationstandardscanberunalongsidetheunknownsamples to allow the DNA concentrations to be estimated. In addition to showing the presence of DNA, the size of the extracted DNA molecules can also be estimated. High molecular weight DNA can be seen as a single band while degraded DNA or DNA that has been sheared during extraction appears as a smear (Figure 4.4). This makes comparison to the standards difficult as the DNA is spread out over a range rather than in a single band. The advantages of agarose gel electrophoresis are that it is quick and easy to carry out and also gives an indication of the size of the DNA molecules that have been extracted. The disadvantages are that quantifications are subjective, based on relative

Figure 4.4
viewing under UV light. The gel shows eight DNA extractions from buccal cells using the QIAamp® Blood DNA extraction kit (Lanes 2-9). Lanes 10-12 contain standards with 25 ng, 50 ng and 75 ng of high molecular weight DNA. A molecular ladder is in lane 1

band intensities; it is difficult to gauge the amounts of degraded DNA as there is no suitable reference standard; total DNA is detected that can be a mixture of human and microbial DNA, and this can lead to over estimates of the DNA concentration; it cannot be used to quantify samples extracted using the Chelex method as this produces single stranded DNA and the fluorescent dyes that intercalate with the double stranded DNA do not bind to the single stranded DNA.

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