- eliana469
Eureka!
When Archimedes got into a bath and realized that the water level rose, he understood that the volume of water displaced should be equal to the volume of the part of his body he had submerged. He then exclaimed that famous expression, meaning "I have found it" in ancient Greek, and run naked through the streets of Syracuse (Sicily) [1]
Making my PCR work did not happened like that at all. First, no one was naked. Second, it was luck.
But, let me just recap.
In the last post about PCR, I talked about the technique, and what we can do to optimize it, when things are not working as expected.
I had received some positive kelp samples for Phaeoviruses from our international parter, the University of Minnesota. I extracted the DNA of those samples, measured its concentration and tried Kelp primers on those: everything worked fine. However, when trying the viral primers on the same samples, my PCR products did not amplify anything. I followed the main troubleshooting techniques [2,3] and still negative results. It was quite frustrating. Again, due to the Coronavirus pandemic, supplies were low and delivery times high.
We strongly believed that there was something wrong with the chemical reaction, but we did not know what. I started reading about PCR inhibitors [4]. I am working with brown algae, which contain polysaccharides that can give you a PCR false negative result. My partners from Bergen, which have experience with brown algae, recommended to dilute my template sample. I tried different dilutions but still no result. I tried cleaning the sample with ethanol, and nothing.
Finally, I decided to spend one week at UiB's lab. I thought they might have a more sensitive PCR machine that would do the magic. It did not. However, the magic appeared after I was given a new Taq Polymerase kit to try! I followed almost the same protocol, volumes and concentrations, but just changed the magical enzyme and the result was what we expected. That is why I say that it was luck. I previously had tried 3 different Taq Polymerase kits, giving all of them the same negative results. The last one was special for hardy reactions, and it did what promised. After making sure the process was already optimized, I started screening my kelp samples. And then is when I found many viral positives among them, and got even more excited.
That is what I am doing at the moment. Screening all my kelp samples for Phaeoviruses. Next step will be to sequence the positive ones.
Finally, I would like to mention the following article about luck in science by Andy Tay: https://www.nature.com/articles/d41586-018-06777-4 . When my PCR worked, I was really happy, but then a dark shadow invade my mind: the solution arrived by chance, it was not the product of a clever mind. Tay argues if we can engineer luck ourselves. Maybe we were lucky at some point of our careers/investigations; but, did we worked for it? Yes, we did. We tried and tried thousand times, we asked many colleagues, we travelled to other labs and tried more things and... finally, we got the result. I still agree that luck exists, and I was lucky to find the proper product that suited my reaction, but I also worked hard for months against demotivation, and got help from many colleagues. So it is fair to acknowledge all of this, and continue moving forward.
Sources:
1. https://en.wikipedia.org/wiki/Eureka_(word)
2. Lorenz TC. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp. 2012 May 22;(63):e3998. doi: 10.3791/3998. PMID: 22664923; PMCID: PMC4846334.
3. Najafov and Hoxhaj. PCR Guru: An Ultimate Benchtop Reference for Molecular Biologists. Chapter 4 - Optimization and Troubleshooting. Academic Press, 2017. Pages 31-43. ISBN 9780128042311.
4. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol. 2012 Nov;113(5):1014-26. doi: 10.1111/j.1365-2672.2012.05384.x. Epub 2012 Jul 24. PMID: 22747964.