Part 2: Teaching Undergraduates to Design an Experiment Before Entering the Lab

Establishing the Scientific Rationale

The first part of the experimental plan asks students to explain the background for the experiment using scientific literature.

I want students to be able to answer two basic questions: Why is this experiment necessary? Why are we using this approach?

This requires more than inserting a few citations. Students need to connect the literature to the experimental decision being made. Doing a literature review is drastically different then developing a protocol and they need to understand all aspects of it.

The plan also asks students to identify any assumptions underlying the experiment. For example, we may be assuming that a protein is expressed in a particular model, that an assay has sufficient sensitivity, or that a treatment concentration will produce a measurable effect without causing unacceptable toxicity.

Students often do not recognize these as assumptions until they are specifically asked to identify them. Making assumptions visible helps us determine whether they are reasonable and whether they should be tested before the full experiment begins.

Defining the Variables and Experimental Groups

Students then identify the independent and dependent variables, experimental groups, and number of replicates.

This sounds straightforward, but it often reveals uncertainty in the experimental design. A student may know what treatment they plan to apply but may not yet have decided exactly what outcome will be measured. They may also confuse technical replicates with biological replicates or select a sample size without being able to justify it.

Writing these elements down gives us an opportunity to address those issues before samples and reagents are used.

It also forces students to consider whether the proposed experiment can actually answer the question being asked. If the dependent variable does not provide meaningful evidence related to the hypothesis, the design needs to be revised.

Understanding Controls Rather Than Listing Them

Controls are one of the most important-and most commonly misunderstood-parts of undergraduate experimental planning.

Students frequently know that they need a positive and negative control because they have heard those terms repeatedly in laboratory courses. However, they may struggle to explain what each control is supposed to demonstrate.

In the planning process, I ask students not only to name their controls but also to justify them.

The goal is for students to understand that controls are not simply additional conditions. They establish whether the experimental system worked and whether the observed effect can reasonably be attributed to the variable being tested.

·       What should happen in the positive control?

·       What should happen in the negative control?

·       What conclusion can we draw if a control fails?

·       Do we also need a vehicle, loading, untreated, or assay-specific control?

Planning the Analysis Before Collecting Data

Students also identify what data will be collected, how the data will be analyzed, which statistical tests may be appropriate, and how the results will be visualized.

I want these decisions considered before the experiment begins knowing full well that the answer may change once the data has been collected.

Waiting until after the data have been collected can reveal that an important measurement was not recorded, that the sample size is insufficient, or that the selected experimental groups do not support the intended statistical comparison. My students always gripe about doing full UV-Vis spectra in the moment, until I ask them for what the rest of the curve looked like and they don't have to repeat the experiment.

Planning the analysis in advance also helps students understand that statistics are connected to experimental design. The type of data collected, the number of groups, the relationship between samples, and the research question all influence the appropriate analysis.

Students do not need to be statistical experts at the beginning of their research experience. They do, however, need to begin asking the right questions and understanding where to get the answers.

Defining Possible Outcomes

One of my favorite sections of the research plan asks students to describe three possibilities:

Students naturally want their hypothesis to be supported. Asking them to define alternative outcomes before collecting data helps separate scientific interpretation from personal preference.

It also reinforces that a hypothesis is a testable explanation, not a result that the experiment is required to produce.

We may also establish go/no-go criteria for moving forward. These criteria define the minimum evidence needed before beginning the next stage of the project. They may be based on control performance, assay reproducibility, sample quality, or the magnitude of the observed response.

Establishing those expectations beforehand makes later decisions more objective.

·       What result would support the hypothesis?

·       What result would refute the hypothesis?

·       What result would be inconclusive?

Anticipating Pitfalls

Finally, students identify potential technical and biological problems.

They may consider reagent stability, equipment limitations, contamination, low signal, sample variability, unexpected toxicity, or limitations of the selected model.

Students will not anticipate every problem, nor should they be expected to. Research is full of surprises.

The purpose is to develop the habit of asking, 'What could prevent this experiment from answering our question?'

When students learn to ask that question before beginning an experiment, they become better prepared to troubleshoot when something goes wrong.

Planning as Part of Doing Science

I do not expect beginning undergraduate students to independently produce a perfect experimental design. The plan is a teaching document, and my level of involvement depends on the student's experience.

Early in their training, students may need substantial guidance. Later, I can ask questions and allow them to identify the necessary revisions themselves.

Over time, the written plan becomes evidence of their growing independence and confidence. Often when a student starts off they are very nervous to try an experiment. They don't want to fail, science is 90% failure though. Teaching them to get used to that while planning for all outcomes helps them be better, more resilient scientists.

The important shift occurs when a student moves from asking me for the next protocol to bringing me a proposed experiment-with a rationale, controls, analysis strategy, and explanation of what we will learn from each possible result.

That is when planning stops being an assignment and becomes part of how the student approaches science.

This is free, always will be — but if you want to say thanks, a chai latte tip is always appreciated ☕

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