Restriction Digestion and Agarose Gel Electrophoresis of Plasmid DNA

The log of a molecule's molecular weight is proportional to the distance that molecule has migrated. Therefore, the first step is to generate a standard curve using molecules of know size (the molecular weight markers).

When using semilog paper (see the next page), the molecular weights (in bp for DNA and kiloDaltons, kDa, for proteins) is plotted on the Y-axis and the distance the molecule migrated is plotted on the X-axis. When generating a standard curve, you will obtain a straight line (use a best-fit line).

Once your standard curve is ready, measure the distance traveled by your molecule of interest. Find that distance on the X-axis, and go up until you intersect with your standard curve. Move over to the Y-axis and that will indicate the molecular weight of the molecule you are studying.

Use the graph paper below and the DNA gel shown to the right to determine the molecular weight of the unknown band indicated with an arrow.

See if this tutorial helps you: http://info.gbiosciences.com/blog/bid/196974/Determining-Protein-Molecular-Weight-with-SDS-PAGE-An-Overview-of-the-Process

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