Sample Preparation for Native PAGE of DNA

Sample preparation for native PAGE is straightforward. Since the DNA does not need to be denatured, one is concerned mainly with buffer content, density and visibility. The salt content of the sample should be adjusted to be no greater than the 200mM salt concentration in the running buffer. Gross imbalances in salt content between sample and gel can lead to salt waves, band distortions and smearing.

The density of the sample mixture is adjusted to ensure that the samples remain in the wells prior to electrophoresis. Generally sucrose or ficol is used for this purpose, as these uncharged compounds will not run into the gel or interfere during electrophoresis. Glycerol should not be used because it forms complexes with the Borate found in TBE buffer. These complexes can migrate into the gel and distort the band pattern.

Finally, tracking dyes are added, typically xylene cyanol and bromophenol blue. These dyes migrate through the gel unsieved. The table below gives the approximate sizes of DNA fragments which co-migrate with these dyes in various percentage 29:1 PAGE gels.

Tracking Dye Co-Migration

Gel % Bromophenol Blue (nucleotides) Xylene Cyanol (nucleotides)
4 95 450
6 60 240
8 45 160
10 35 120
12 20 70

 

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