Agarose gel electrophoresis uses electricity and a porous gel matrix to separate mixtures of DNA molecules into discrete bands based on their size. As the current is running, the DNA molecules move through the pores in the gel. Since it’s easier for small molecules to move through the gel than larger ones, the DNA separates into bands by size, with larger molecules near the wells, and smaller molecules further down the gel.
Mathematically, we say the distance travelled is inversely proportional to the length of the DNA fragment – larger fragments move a smaller distance. However, the speed that each fragment travels is not equal. Small molecules move much faster than larger ones, creating a non-linear relationship between size and migration. Therefore, the migration distance is inversely proportional to the log base 10 of fragment length.
We can use this relationship to determine the size of DNA fragments in the agarose gel. On each gel, we run a DNA ladder alongside our experimental samples. The ladder contains a series of DNA fragments of known molecular weight. (To learn more about a DNA Ladder, be sure to check out our video on YouTube or our previous blog post.) We measure and plot the migration of each band in our DNA ladder and add a best-fit line. Once complete, this standard curve lets us calculate the size of each experimental sample.
Building your first standard curve can be a challenge – there are some tricky parts! To help you build your first standard curve, we created a new video tutorial! We also have a blog post that describes building a standard curve.