Overview DNA sample is mixed with DNA loading dye prior to loading into the wells of agarose gel. DNA loading…
A 10 ml of 6X DNA loading dye can be prepared by dissolving and mixing 40 mg orange G, 25 mg xylene cyanol FF, 1.5 g Ficoll 400 and 0.1 ml of 1M Tris.Cl, pH 7.6 in sterile deionized or distilled water to a final volume of 10 ml.
Migration of Bromophenol Blue and Xylene Cyanol in Agarose Gel Running in TBE or TAE Electrophoresis Buffer
Bromophenol blue and Xylene cyanol are the two most commonly used tracking dyes for the analysis of DNA on agarose gel electrophoresis. These negatively charged dyes not only help in monitoring the progress of agarose gel electrophoresis but also allow easy monitoring of sample loading process onto the wells of agarose gel. Their position in relation to DNA fragments is an important information that helps to determine how far a gel must be run without accidentally letting the DNA fragments of interest run out of the gel and at the same time ensuring good resolution among different size DNA fragments.
DNA sample is mixed with DNA loading dye (also called sample loading dye) prior to loading onto the wells of agarose gel for electrophoresis. DNA loading dyes contain a high-density reagent and tracking dyes. DNA loading dye makes the DNA sample heavier and coloured, thus helping the DNA sample to sink into the agarose wells without diffusing out and enabling us to monitor the loading process. In addition, tracking dyes in loading dye migrate as a diffuse band to the same direction as DNA which can be seen visually thus allowing us to monitor the progression of electrophoresis. However, tracking dyes sometimes interfere in the analysis of DNA bands by masking it.
10 ml of 6x DNA loading dye containing Bromophenol blue, Xylene cyanol FF, and Ficoll 400 is prepared by dissolving 25 mg bromophenol blue, 25 mg xylene cyanol FF and 1.5 g Ficoll 400 in water to a final volume of 10 ml. The final solution (6x DNA loading dye) will contain 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF, and 15% (w/v) Ficoll 400.