A Miscalculated Step
As a graduate student, Tharin Blumenschein learned that too much sodium hydroxide breaks down more than just bacterial cells.
Mariella is an assistant editor at The Scientist. She has a background in neuroscience, and her work has appeared in Drug Discovery News and Massive Science.
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In the mid-1990s, I was a graduate student at the University of São Paulo working with EF-hand proteins, which are regulatory calcium and/or magnesium binding proteins characterized by two α-helices (E and F). I wanted to understand which amino acids in the protein’s binding site determine its affinity and specificity towards those ions.1
To explore this question, I transformed mutated EF-hand sequences into bacteria using plasmids to obtain larger plasmid amounts for my protein expression experiments.
One day, I was getting ready to purify the plasmids from the bacterial cultures. Since we did not have kits to isolate these circular bits of DNA back then, I had to prepare all the solutions in the protocol, including one that uses sodium hydroxide to break open the bacterial cells. After lysing the bacteria, I added cesium chloride (CsCl) and ethidium bromide (EtBr) to my bacterial lysates. CsCl creates a gradient that separates the plasmid DNA after high-speed centrifugation.
When my centrifugation time was up, I went to the equipment with syringes to collect the plasmid DNA from the tubes. Once I pulled the tubes out and looked for EtBr-stained DNA bands, I was surprised to see no bands.
Intrigued, I repeated the purification process, but it did not work. After confirming I didn’t miss any step in the protocol, I decided to redo my calculations for all the solutions. That’s when I realized that I had added ten times more sodium hydroxide to lyse my bacteria!
After overcoming my initial disappointment at making the mistake, I repeated the protocol with the new lysis solution and, lo and behold, I was able to obtain the plasmid DNA I needed.
This experience reinforced the importance of double-checking calculations before moving forward with an experiment. It also taught me an interesting property of sodium hydroxide, which can break down DNA molecules when used in excessive amounts.
This interview has been condensed and edited for clarity.
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- Blumenschein TM, Reinach FC. Biochemistry. 2000;39(13):3603-3610.
