Simple, cheap loading dye for Agarose gel electrophoresis
|Summary: An Agarose loading dye should be dense (so your sample falls into the well when you load it), dark when you load it (so you can see your sample), faint when you image it (so you can't see the dye spots in your image), and cheap. Such a dye can be easily made by diluting Fisher 5x Sucrose Dye (Part No: BP655) 1:20 into 40% sucrose.|
Article created: Sept 14, 2007
Last update: Sept 15, 2007
Article by: Jeremiah Faith
First (main) purpose of Agarose gel loading dye to add density to your sample
This density prevents your sample from floating out into the buffer after you load it into the gel. The dye is typically in a viscous solution such as glycerol or sucrose to make it dense. Even with the loading dye, you should try to load and run your samples relatively quickly or the samples will start to diffuse out of the wells anyways.
Second purpose of Agarose gel loading dye to add color to your sample
With this added color, you can tell if you are accurately pipetting your same into the well or if you are clumsily losing your entire sample by accidentally pipetting it into the buffer. The color also allows you to roughly track where certain DNA fragment lengths will be on your gel as the different dyes move along the gel at different rates. What length in bp your dye roughly tracks is dependent on the dye and on the percentage of the gel.
Sometimes lab beginners incorrectly believe that the colored dyes you see are actually the stains that allow you to see the DNA. The dye does not stain your DNA and has little influence on the DNA in your sample. However if you use huge amounts of dye (not recommended and not useful), your DNA and the dye will move through the gel a little slower than other lanes with less dye.
My approach to gel loading dye I don't waste time weighing and mixing dyes for my Agarose gel loading dye. The dyes are extremely messy, will stain your precious lab coat (or your clothes if you're clever enough to mix your dyes without a lab coat), and are only slightly cheaper than the method I use.
I buy the 5x sucrose loading from Fisher (Part No: BP655). If you use this dye at full concentration, you'll have nasty dark dye spots when you image the gel. This extremely dark dye phenomena is pretty uniform across all commercial dyes I've tried. But as I mentioned above the main purpose of the loading dye is not the color, but rather the density.
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