By John Brennan
Updated Mar 24, 2022
Gel electrophoresis remains the workhorse of molecular biology laboratories. By applying an electric field, DNA and proteins are separated—usually by size—within a gel matrix. Although the technique is common, the way results are interpreted varies with the downstream application, whether it’s a Western blot, Northern blot, Southern blot, or simple agarose run.
When you’re working with agarose gels, two tasks dominate: 1) distinguishing uncut, nicked, and linear plasmids from your insert, and 2) estimating fragment sizes using a reliable standard curve. The following steps walk you through that process in a clear, reproducible manner.
Consult your lab notebook to confirm which sample was loaded into each lane. The lane labels you recorded at the time of loading are your starting point for all subsequent calculations.
The ladder contains fragments of known length. Its migration distances will serve as the reference for sizing your unknown bands.
Using a ruler, measure the distance from the well to the tracking dye (the dye that travels the farthest). Record this value; the units are arbitrary as long as they are consistent.
Measure each ladder band’s distance from the well, then divide by the tracking dye distance. This yields the relative mobility (mobility factor) for each standard.
Example: If the tracking dye traveled 6 inches and ladder bands traveled 5, 4.5, and 3.5 inches, the relative mobilities are 0.833, 0.75, and 0.583.
Enter the relative mobilities and corresponding fragment sizes (in kilobases) into a spreadsheet. Manufacturers typically provide these sizes in the ladder’s datasheet.
Create a graph with relative mobility on the X‑axis and fragment size on the Y‑axis.
Use the Trendline feature to fit a power‑law equation (e.g., y = ax^‑2). Aim for an R² of at least 0.90 to ensure a reliable curve.
Smaller fragments migrate farther. Note that supercoiled (uncut) plasmids travel farther than linear fragments of the same size, while nicked plasmids migrate slower.
Cross‑reference each lane’s bands with the sample you loaded. For a plasmid digested with two enzymes, you should see two distinct bands: one near the top (insert) and one near the bottom (cut plasmid). A single‑enzyme digest should produce a single band that travels slightly farther than the uncut plasmid but far less than the insert.
Measure the distance from the well to each unknown band, divide by the tracking dye distance, and obtain the relative mobility.
Insert the relative mobilities into the equation derived in Step 7 to calculate the approximate size of each fragment.
If you observe broad, bright bands near the bottom of every lane, you likely have RNA contamination—review your purification steps.
