My First Gel Electrophoresis Run

My first gel electrophoresis run! Actually, strictly this is not true as I did one last year for our assessed practical*, but the gel was already pre-prepared and from what I remember we only really pipetted our samples into the wells while the rest was more or less done for us.

In this example we're determining the identity of two unknown dyes by comparing the bands produced by gel electrophoresis to the 8 other known dyes also in the run. The gel was prepared as a 0.8% concentration of agarose with TAE buffer and run at 90 volts for half an hour. I've included a summary of the results below.

Fig1. Substances in wells 1-10 are seen migrating either towards the anode (red), the cathode (black), or staying still (neutral). Lines A, B, C, and D act as a ruler for the 4 bands formed by dyes 9 and 10. A starting point “line X” has been included for comparison.

Fig2. Table of dyes, their respective well numbers, and the charge as derived from the movement towards anode or cathode as depicted in Fig1.

Based on the intersections of lines A (with 1 and 9) and D (2 and 9), the unknown dye in lane 9 is a mixture of bromocresol green and methylene blue.

Based on the intersections of lines B (6 and 10) and C (4 and 10), the unknown dye in lane 10 is methyl orange, and safranine orange.

The most remarkable observation is that Line D shares its distance not only with dyes 2 and 9, but also 3. Although it becomes clearer when comparing colour that the band formed by (unknown) mixture 9 is homologous with dye 2 (Bromocresol Green), the fact that the purple dye #3 (Bromophenol Blue) has also run to the same distance implies that Bromophenol Blue has a similar size, density and charge to that of Bromocresol Green. This appears to be the case as they have very similar molecular structures, differing only in the addition of 2 methyl groups, as laid out in Fig3 below.

The shearing on bands 1, 6, 7, 8 and 10 may be due to either overly concentrated dilution of dyes, too great a volume of dyes being pipetted resulting in overfilled wells, or running the gel too quickly. Difference in concentration could be tested for by using spare empty wells to load 50% dilutions of the dyes with the biggest trails (eg. 6 and 7) and compare their trails with their concentrated counterparts as part of the run. Similarly, the gel could be run on a diminished voltage to see what affect this would have on the quality of the run.