Answers for control experiments

BIOC/MCB 568 -- Fall 2009
John W. Little--University of Arizona

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The web page on Control Experiments asked what hypotheses could explain the three likely outcomes of a DNA transformation experiment. Here are some hypotheses:

a. A few hundred colonies appear on the plate. A likely (and desirable) hypothesis is that these colonies result from the growth of single cells that took up the plasmid DNA, which made them antibiotic-resistant and hence able to grow in the presence of the antibiotic.

b. No colonies appear on the plate. A likely explanation is that your cells are not "competent", that is, they cannot take up the DNA efficiently.

c. A very large number of colonies appear. Two likely hypotheses are that your cells are already resistant to the antibiotic, or that your plates do not contain the antibiotic.

You were then asked to identify alternative hypotheses, and develop controls to distinguish between these hypotheses and the ones given above.

Outcome a: Another hypothesis is that your competent cells contain a small number of antibiotic-resistant cells. The proper control to test this hypothesis is to have another transformation to which you don't add plasmid DNA. Why is this a good control? Think about what each hypothesis predicts. This is often termed a "negative control".

Outcome b: Other hypotheses are that your DNA is bad (e.g., it is degraded), or it does not confer resistance to the antibiotic, or that the plates were made wrong. One control likely to be helpful is to use a DNA preparation that you know is good (that is, it has been shown to give transformants in other experiments); this is often termed a "positive control". To test if the plates were made wrong, use a different batch of plates.

Outcome c: Either hypothesis above probably means that you will have to repeat the experiment, probably making a fresh batch of plates and getting more information about your plasmid. This outcome is not very common, and controls are not generally included to take it into account.

Back to page on Control Experiments.