Agarose Gel Electrophoresis

Summary of Activity Goals

Gel electrophoresis is a powerful technique in molecular biology that enables separation and visualization of biomolecules such as DNA, RNA, or proteins. The main goals of this activity include introducing how gels work, showing what kind of data can be acquired using gels, and how gel data can be interpreted as part of a biological experiment. The activity makes use of a simulation tool, Gelbox, that allows users to quickly explore how changing experimental parameters can affect the data output from gel electrophoresis.

Summary

The goal of this exercise is to reinforce the students’ understanding of how different nanoscale physical characteristics of molecules are manifest as macroscale band patterns in gel electrophoresis data. The students will be provided with descriptions of several different gel sample runs and will need to predict what the resulting gel data will look like.

Content

Gel electrophoresis is a very powerful tool for isolating, defining, and characterizing biomolecules such as DNA, RNA, and proteins.

However, it is important to keep in mind that just because two gel bands migrate at the same speed and intensity does not mean that the molecular species are necessarily identical.

The gel pictured here was created using the Gelbox simulator. It includes the default "1kb ladder" sample in the first lane, and five additional samples.

Open the gel box simulator, which can be downloaded at http://douglaslab.org/gelbox/. Use the simulation to try and answer the questions below

Click here to view the simulation.

Can you describe at least five samples that could migrate at approximately the same speed but in fact are different at the molecular level?

• A. 3000 bp monomer (100 ng)
• B. 1500 bp dimer (100 ng)
• C. 1000 bp trimer (100 ng)
• D. 3800 bp elongated molecule (e.g., supercoiled) (100 ng)
• E. 3000 bp monomer (50 ng) + 3000 bp monomer (50 ng)

Exercise 2: The limits of the Luria-Delbruck experiment.

Summary

In this exercise, students will apply their knowledge of gel electrophoresis to help "troubleshoot" some experiments that yielded unexpected results. The student will read a description of an experimental setup, for example, a DNA sample of a given length that was cut at a certain site with an enzyme. The student will need to interpret various gel images to determine what part of the experiment did not work as expected. For example, given a gel image that shows a single band corresponding to the length of the uncut DNA, the student could suggest several possible explanations (the enzyme was accidentally omitted, the DNA sequence may be incorrect, etc.)

Content

When an experiment yields unexpected results, scientists often must consider whether the root cause is poor experimental design, user error, or a truly novel scientific result.

Open the gel box simulator, which can be downloaded at http://douglaslab.org/gelbox/. Use the simulation to try and answer the questions below:

Click here to view the simulation.

Consider the following simulated gels based on the samples from the previous exercise. Provide an interpretation of what might have caused the result. Describe what, if anything, you might do to fix the problem.

Answer Key 2

• A. All bands are heavily distorted, indicating that the gel was probably cast with only water (H2O) as the buffer instead of TBE or TAE. To fix, prepare another gel and make sure to include the correct buffer.
• B. Most of the expected bands are missing, and the remaining ladder bands migrated up instead of down, indicating that the polarity of the power source was reversed. Check the power supply and ensure the gel apparatus wires are matched to the correct terminals.
• C. It appears that most of the samples have run off the end of the gel. Perhaps the gel was likely run for too long or at too high a voltage. Repeat the experiment with a decreased duration and/or voltage.
• D. No samples are visible on the gel, including the ladder. The gel may have run backward (similar to B, but for longer).
• E. The ladder looks good, but the samples are faint. Try increasing the concentration and/or volume of loaded samples.
• F. Samples are present, but the ladder is missing. Re-run and ensure ladder is present.
• G. The ladder is present but samples are missing. The samples may have floated out of the wells - check for residual ethanol, or supplement the sample with an additive to increase density (e.g., tetraethylene glycol). Revisit experimental design and possibly add additional control samples.
• H. Bands are slightly distorted/wavy. This may be caused by physical damage to the sample wells, for example, due to pulling the gel comb out too early before the gel has completely solidified. Check for high voltage and consider possible salt effects.