This week, I needed to collect data on PCR amplification of the gram-negative species used for my study. Amplicons were detected post-PCR in multiple previous experiments; however, over the summer an anomaly arose after a PCR when two of the targeted species failed to amplify. This anomaly caused me concern, so I decided to re-test to see if the negative result could be repeated.
The original DNA samples I used for that PCR have degraded, so I had to perform new extractions. This is not optimal, of course. I would have preferred to re-test all of the original components, as it would be the only certain way to control the experiment, but too much time had passed since the original extraction. Additionally, I did not have enough of the original primer dilution to re-test all nine species.
From inception, the re-test was not a repeat of the original conditions that resulted in the anomaly. So while with new ingredients I could attempt to narrow down the reason for the amplification failure, there was too much variability from the summer procedure to be certain as to the cause.
This was borne out by the new PCR I ran. For the first time testing the set of PCAT-4 primers I designed for this study, amplification was unsuccessful on five of the nine tested species. I am stymied as to the reason why; there are simply too many variable changes to immediately ascertain a cause. I will have to repeat the experiment, changing one variable at a time, until I isolate the reason for the results.
That was my week. I have not had negative results for some time, and I was a little surprised (possibly even vexed) by the results. But as a wise wag (Josh James) once told me, "In science, there are no wrong answers, only new directions to explore." So I will simply explore those new directions and go where the science takes me until I find the answers I am looking for.
Cheers! See you in the lab.
PS: I just ran across a study that purports that humans emit a personalized microbial cloud that can be used to identify the unique individual. Researchers measured microbial emissions from a series of individuals and then used 16s gene sequencing to identify bacterial contributions from those subjects. It's worth a read, so I have included a link here to the published study. Enjoy!
PPS: One of the species I am targeting for amplification, Serratia marcescens:
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