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.
- 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.
- 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.)
- 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 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.
- 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?
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