Agarose gel electrophoresis is an important tool for visualizing DNA. To be more precise, electrophoresis, not always done across an agarose gel, is the only tool for visualizing DNA. Both high school and college biology students gain an intuitive understanding of the nature of DNA and other proteins by working with electrophoresis, one of the most important teaching tools available from Modern Biology, Inc.
With electrophoresis teaching tools from Modern Biology, your students will gain the practical skills they need to master advanced electrophoretic methods. With Modern Biology, your students will learn through experimentation, not just from demonstration. Your students will make statements of expected results, and then compare their results to their initial hypotheses. But sometimes unexpected results are the best learning experiences.
What factors affect the outcome of electrophoresis?
There are a number of ways that experimental technique can affect the outcome of electrophoresis:
- The composition and concentration of the buffer. Students need to understand how pH of the buffer affects the movement of DNA and proteins in response to electrostatic charge. When they don’t get the expected motion of test and control substances across the gel, they need to be able to account for differences in terms of pH, if their buffer was not mixed to a standard pH.
- The concentration of the agarose gel. Not following instructions for preparing the agarose gel won’t necessarily affect the relative rates at which test substances and controls travel across the electrostatic field. But teams that don’t prepare the gel properly may get results much sooner or much later than others in the class.
- The purity and concentration of the DNA. Different molecules travel at different rates. If the DNA sample is contaminated, students will not see clear bands forming across the gel. Students need to understand how purity and concentration of DNA affect the precision of the bands that form on the gel, or the lack of it. Contaminated DNA, RNA, or other test proteins will produce a smear.
- The voltage of the electrophoresis gel. Choosing the right voltage is critical to the success of electrophoresis. The higher the voltage, the faster molecules will move. But lowering the voltage for slower movement of molecules will result in better definition of the bands. Voltage that is set too high can melt the gel.
- Use of the buffer and agarose gel. Damage to the cells in the electrophoresis plate may prevent proper migration across the gel.
- Preparation of the gel. Test substances can’t travel through a bubble in the gel. Failure to mix the gel properly may mean that one of the controls that creates the DNA ladder or the DNA sample itself is not read accurately.
- The pH of the buffer. Buffer solution ideally has a pH of about 7.0. If the pH of the buffer is too far below 7.0, both test materials and controls will migrate across the gel faster than expected. Letting the process run too long will invalidate the DNA ladder at the side of the gel used to estimate the size of the molecule. On the other hand, if the buffer is too alkaline, if its pH is too far above 7.0, the migrations across the gel will take too long and students may run into time issues. This problem can undermine the confidence of students while they are learning the technique, so it is important to provide students with distilled water and tools for monitoring pH before they add it to the electrophoresis cells.
Understanding what can go wrong with electrophoresis won’t prevent students from having to re-run the experiment to get a good read of the results. But knowing what went wrong also helps students know what went right, and where they can correct inevitable problems with technique to get better results next time.
Modifying Instructions for Modern Biology Experiments to Teach Principles of Electrophoresis
Modern Biology’s electrophoresis experiments coordinate reagents and equipment, so your students will have maximum opportunities for success. Following instructions to the letter will bring expected results.
But when students don’t follow instructions to the letter, they need to understand the results of their deviations from prescribed technique.
These experiments include a Time and Voltage table in the instructions for ease of use. These instructions tell students how long DNA fragments should be subjected to electrophoresis to separate them.
But when time gets away from a class, it’s important to remember that longer exposure to voltage differentials results in greater separation of lines of dyed DNA only up to a point. If the voltage is applied for too long, all the DNA samples will migrate to the other side of the gel, and all information will be lost.
When students expect that their test sample will have a length very similar to a control sample, they should run the electrophoresis as long as possible to get maximum definition between bands. But to make sure that samples don’t begin to collide at the positively charged side of the gel, students need to slow down migration.
How?
- The more concentrated the gel, the more slowly fragments will move, and the better definition students will get between bands. DNA samples migrate more slowly in a 2.0% gel than in a 1.0% agarose gel.
- The thicker the gel, the more slowly the DNA fragments will move. A gel that is 8 mm thick will slow down molecular motion more than a gel that is 6 mm thick. Students need to be able to calculate how many millimeters of solution are needed for a given thickness of gel.
Another important point:
Voltages may vary in older electrophoresis units. Paying close attention to the migration of dyed DNA is enough, however, because DNA fragment size is determined relative to known standards. As long as the electrostatic charge across the gel is the same along the gel, fluctuating voltage will not be a problem. But this means some pairs of students may get results faster or slower than their peers.
