• Helen

Extractions, Runs, Extractions, Runs, Extractions

My time at UFZ has been mostly consumed with differing forms of extraction methods followed by further testing via qPCR. For the first couple of weeks in the lab I was finishing up the final extractions for the Bd/Bsal swabs from the Pyrenees that were taken during the summer of 2016 and 2017 and then set up these extractions in multiple qPCR runs. This is when I encountered my first frustration.

Science is full of these moments where your method works perfectly and then something happens and it no longer does! On a Friday the Bd/Bsal qPCR duplex I was running worked perfectly, the standards were amplifying and I was seeing results. On the next Monday afternoon I resumed my day of qPCR runs only to find that the Bsal standards were not amplifying correctly and the results were all over the place. I left for a weekend and came back to disaster! I’m being dramatic, but it was frustrating to have a method be working so well one day and to it suddenly not the next. So that led me on almost a full week of testing. Did I not prepare the primers correctly? Is the probe bad? Did I screw up the standards? Answers: Nope, no, and turns out not. That left the machine… Monday morning it had been re-calibrated prior to my first runs. Had the re-calibration somehow messed with reading the Bsal probe? Absolutely. Tests on a new machine confirmed that I was not a fool in the lab and it was the machine and not the method that let me down. Oh, the woes of science.

An ugly amplification plot.... source of frustration.

Following the Bd/Bsal testing, I moved onto Ranavirus. Unlike the simple Prepman extraction method I was using for the Bd extractions, the Ranavirus samples required extraction using a kit entitled DNeasy Blood and Tissue, which while it is straightforward and in fact easy to follow, it was one of the more boring and time intensive extraction methods I’ve worked with. The qPCR runs for these extractions were simple enough the first time around. The results followed the populations and seemed to make sense. However, when I went to retest the positives, I could not get all of them to amplify again. Hence started what I have called Helen’s Frustration Week #2 at UFZ. The reason for these inconsistencies is still being figured out.

Generally nice standard curves and some positives, but not all samples amplified all the time... another source of frustration.

And my latest task: extracting the skin microbiome samples. While just as time consuming as the DNeasy extraction method, the NucleoSpin Soil kit is at least more entertaining. And testing the total DNA of each extracted sample via Qubit shows there’s DNA in each sample! I’m doing something right. But the DNA is of what, exactly? Not sure yet. Answers will come later on as I continue with my microbiome processing. Stay tuned!

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