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Gel Electrophoresis

High schoolBiologyAn illustrative log-size agarose migration calibration with polarity, band spreading, run-off and a visible-ladder regression for size estimation

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About this experiment

Load several DNA fragment mixtures into a gel and follow their separation. Use the size ladder as a measurement reference rather than reading hidden sample labels. Compare polarity, run time and gel concentration, then examine why closely spaced fragments can overlap or leave the gel.

Learning goals

  • Relate smaller DNA fragments to farther forward migration.
  • Estimate fragment size from a log-size ladder calibration.
  • Investigate polarity, excessive run time and limited band separation.

How to use it

  1. 1Load the selected lane with comma-separated fragment sizes and switch on power.
  2. 2Run for thirty modeled minutes and compare sample bands with the ladder.
  3. 3Load unknown samples, click a sample band and inspect the ladder-based size estimate.
  4. 4Compare reversed polarity, higher gel concentration and a longer run, recording bands that leave the gel.

The model

Prescribed migration position in centimetres
Inverse visible-ladder calibration

Assumptions

  • DNA is negatively charged and moves toward the positive electrode. The gel is 8 cm long with wells at 0.6 cm; the selected voltage is normalized to a 100 V reference.
  • Band position uses a prescribed linear relation with log10 base-pair count, voltage, time and gel concentration raised to 0.8. This calibration is illustrative rather than an experimental protocol prediction.
  • Band spread is a Gaussian width estimate increasing with time. Intensities are false-color markers without a DNA-mass model. Size estimation fits position against log size using at least two visible ladder bands; off-gel bands are excluded, and interpolation is restricted to the visible ladder range.

Try this in class

  1. Q1Why can very similar fragment sizes remain unresolved despite different exact positions?
  2. Q2Why can a longer run reduce the usefulness of the size ladder?

Frequently asked questions

Are the hidden sample sizes guessed by an AI model?

No. A deterministic regression uses known visible ladder sizes and their positions.

Why does size estimation disappear before running or after all ladder bands leave?

Zero migration gives no size-dependent slope, and fewer than two visible reference bands cannot define the calibration.