So I have been spending my days standardizing DNA extraction protocols. During my PhD days, I worked with rich agricultural soils. These soils were rich in organic matter and nutrients. Extracting quantifiable microbial DNA from such systems is easy. Things are however a little different now. I am working with systems that are essentially oligotrophic in nature, with very low carbon and nitrogen inputs. Extracting quantifiable DNA is a challenge since microbial counts in these systems are low to begin with. I have been working with some mine tailing samples from the Maier lab as well as trial extractions from miniLEO soil. I have also been testing out some heterotrohic plate count experiments and trying out kits from different manufacturers. This is also a good exercise as I am timing myself. Right now, I have progresssed from taking 5 hours to extract DNA from 4 samples to 7 hours for 12 samples. I also need to plan this out since I am mentoring an Honors intern and am responsible for his project deliverables. He is working with me on the miniLEO samples and we need to have a realistic idea of how many days we are going to take to extract DNA from a representative set of samples from miniLEO. We have decided on a total of 80 samples. 10 samples a day implies just over a week's work!
Having enough extractable DNA also has implications for future Illumina MiSeq experiments which I am planning to do. A minimum of 50 picograms/ul is required as optimal DNA concentration for amplification prior to sequencing. Right now, with about 1.2 g of mini LEO soil, I am able to achieve just over 50 pg for most samples. I may have to revisit some samples with low counts. However, things look promising so far!
And here is a set of extraction tubes (the blue color is Brilliant Blue dye that was added to miniLEO to track hydrological flow patterns in the hillslope). Also, I plated out the basaltic parent material and three regions from the top most layer of miniLEO. The plates are pretty interesting to view! I also taught my intern to collect soil samples from around campus, plate the solutions out, and extract microbial DNA from those samples! Time to start working with some real samples. Trials are over!
