Gel Electrophoresis:
Getting It Right

Why Gels Work

Gel electrophoresis separates molecules by size using an electric field and a porous matrix. Nucleic acids are negatively charged (thanks to the phosphate backbone), so they migrate towards the positive electrode. Smaller molecules move faster through the pores of the matrix, larger molecules get held back. The result is size-based separation, which you visualise with a stain or a fluorescent dye.

The principle is the same for both agarose gels (nucleic acids, typically) and polyacrylamide gels (proteins and small nucleic acid fragments). What changes is the matrix, the pore size, and therefore the resolution range.

Choosing Your Agarose Percentage

The concentration of agarose determines the pore size and therefore the resolution range. A 0.8% gel resolves large fragments well (1 to 10 kb range) but smaller fragments run off the end. A 2% gel resolves small fragments (100 to 500 bp) but large fragments barely enter. The percentage you choose depends entirely on what you are trying to resolve, and getting this wrong is one of the most common reasons people cannot see their band or cannot distinguish two bands that are close in size.

For most routine work, a 1% gel is a reasonable starting point. But if you are trying to confirm a restriction digest with fragments that differ by less than 100 bp, you need to go higher. If you are checking genomic DNA integrity, you need to go lower. The choice is not arbitrary.

Loading Dyes and Migration

Loading dyes serve two purposes: they add density to your sample so it sinks into the well, and they provide visible markers so you can track how far the gel has run. Bromophenol blue and xylene cyanol are the two most common tracking dyes, and they migrate at different apparent sizes depending on the gel type and percentage. Knowing where your tracking dye runs relative to your fragment of interest is important, because if you run the gel until the dye reaches the bottom, you need to know whether your fragment has already run off.

Ladders

A molecular weight ladder is your ruler. Without it, you are guessing. Choosing the right ladder means matching the size range to your expected fragments. Running a 1 kb ladder when your fragments are all under 200 bp is not particularly informative. Similarly, prestained protein ladders vary in their mass-per-band loading, their colour patterns, and their coverage of the molecular weight range you care about.

What BenchCalc Does

BenchCalc includes an agarose gel calculator for pouring gels with TAE/TBE buffer selection and stain volumes, a PAGE gel calculator for SDS-PAGE and native gels, and a reference section with 13 gel ladders (6 protein, 7 DNA) with band sizes, prestain colours, mass per band, and loading volumes. It also includes dye migration tables for bromophenol blue and xylene cyanol across different gel types and percentages.