How Much Control is Enough?

The purpose of a control is to allow you to fully interpret all of your results. What often happens is an experiment is designed and successfully executed only to fall short due to inadequate controls. These results go into your notebook but will never be seen in public. You are too embarrassed to admit you left out the one control condition that would have made your experiment the brilliant example of the scientific method you originally intended. However, it is possible to have too many controls; there is no need to control for every single variable (color of your socks, for example), only the variables which have the potential to influence the interpretations of your results.

Below you will see some results that are just short of being publishable (i.e. wasted effort). Your mission, should you decide to pass this course, is to interpret the results fully. This means you should give all reasonable explanations of the results (no need to mention phase of the moon etc.). Then, redesign the experiment to include the controls necessary to distinguish between all the explanations you listed for the original data. Situation number 5 is a bit different, so you will be asked to do a different task.

1. You have done a ligation of a 2.3 kb fragment into a plasmid. You know that the gel purification worked because you ran some purified DNA on a gel before you attempted the ligation. After the ligation, you transformed the ligated DNA into some E. coli and plated the cells onto a LB (standard growth medium) plate and you see a lawn of cells. There are 2 possible explanations and at least one very important control.
   One possibility is that all of these cells took up the DNA (not likely). The other is that since you plated the cells on LB, you did not have a basis for positive selection of transformants. Therefore, the control you should have run is a mock transformation - no plasmid DNA. You would expect no colonies to form on this plate.
2. You have just spent the last 5 days working on a Southern blot and you very excitedly go into the dark room to develop the X-ray film. Loaded onto the gel was one lane of molecular weight markers and five lanes of genomic DNA from five endangered species, so you were very careful with the samples. These species had never had their DNA probed (with a fly cDNA) before and you were the first in the world to do it. You process the film and stagger backwards at what you see - a blank film. You were so excited about this blot that you had scheduled a meeting with your thesis advisor and now you have a lot of explaining to do - get busy. Give all probable interpretations and redesign your experiment. (The second moral of the story is don't schedule the meeting before you see the blot.)
   Since you did not have a positive control, it is impossible to determine if the probe was good or not. So, you cannot interpret these results and now the World Wildlife Fund is angry at you. So, next time, put some fly genomic DNA on the blot too. This will serve as a positive control.
3. You have cloned the single gene that could cure all cancers (congratulations) and you decide to call the gene FAR (Fame And Riches). You want to show that FAR is not expressed in normal cells but is expressed in all cancer cells. You transfect this FAR DNA into a human cell line and immunofluorescently label the expressed protein (you made a monoclonal antibody against FAR in your spare time). What you see is bright labeling in the cytoplasm. Then you look at some immunofluorescently labeled sections from normal and cancerous tissues and you see that all cells are brightly labeled in the cytoplasm. Interpret these results. What controls should you have included with 1) the transfected cells experiment and 2) the tissues experiment?
   You should have done a negative control when you tested you antibody on the transfected cells - cells that had not been transfected. If this had been labeled with your mAb, you would have realized earlier that your mAb is not specific for FAR. Therefore, you cannot fully interpret these result. Perhaps FAR is expressed in all cells but is mutated in cancerous cells but not in healthy tissue (similar to ras ). Alternatively, your antibody is not specific to FAR and binds to another common protein. Too bad.
4. You want to clone the mammalian gene that encodes the first enzyme in the pathway that allows us to metabolize glucose. To do this, you decide to utilize a yeast strain that is leu2- and is defective for this same glucose catabolic enzyme (this mutant strain grows fine on fructose + leucine but cannot live on a glucose + leucine medium). You isolate human genomic DNA and ligate it into a 2 µ yeast shuttle vector that also has the LUE2 marker. You electroporate the ligated plasmids into yeast and plate the cells onto a glucose plate, but no cells live. What went wrong?! This should have worked, you know humans encode this protein too. Explain these results and repeat the experiment with good controls.
   You forgot to do this in stages. You should have plated the electroporated cells onto fructose plates to select for transformants, and then replica plated these transfromed cells onto glucose plates. As it is now, you do not know if the transformation worked or not. By doing it in steps, you can verify that the transformation worked, and then go looking for the gene by complimentation.
5. Judging from the four examples above, it has been a difficult year for you. You have switched fields 4 times because you have been a little careless. This time you will get it right. With your usual good luck, you think you have cloned a gene that will prevent the common cold (this time you call it RID, Rolling In Dough). You cloned the yeast RID gene that prevents yeast from catching colds. You then decide to clone the human homolog by using the yeast cDNA as a probe to screen a human genomic library. You plate the library, screen it and find one positive plaque. You rescreen this phage and it is positive again. You are very satisfied with yourself, lean back, open a brew, and reach for the phone to call the New York Times. Abruptly, you hang up. You decide that you have not done one very important experiment. What should you do before taking dancing lessons so you can dance with the queen at the Nobel awards ceremony?
   You have not determined if this gene is functionally responsible for the phenotype you want. You should express this human gene in cells that can catch colds (rid- yeast cells in this fictious world). If this human homolog can protect the yeast cells, then you know you have gotten it. But always verify your clones with a functional assay if possible.

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