Now that DNA has been extracted, I can move on to quantifying how much DNA is present in each sample and then how much of a particular gene is present in each sample. To quantify the total DNA concentration, we use a Qubit. The Qubit assay includes a dye that binds to DNA and then the Qubit detector measures the amount of dye bound to DNA. Once I know the total DNA concentration for each sample, I have dilute each to the same level before doing quantitative Polymerase Chain Reaction (qPCR). For qPCR, we choose a gene of interest, often the 16S rRNA gene, and we amplify that gene from every single organism that has it. We can use a dye (similar to the Qubit) to learn how much the gene amplified and backtrack how many genes were there to start. This is helpful because you can learn much more about which organisms are in the sample and how abundant they are without having to do the more intensive sequencing process. In my case, I want to know how many cyanobacteria are in the soils. It is really easy to visually estimate the abundance of mosses and lichens at the soil surface, but it is much harder to accurately estimate the number of cyanobacteria. That is why qPCR comes in handy!
I am doing all of these steps for multiple different projects. In some cases, I won't do anything else with the DNA. The qPCR is the end-point of the analysis. In other cases, I will use the qPCR data to determine which samples to sequence (which samples are worthwhile spending the additional time and money to learn about the microbial communities in depth).
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AuthorSierra is a graduate student in the Barger Lab at CU Boulder studying microbial ecology for dryland restoration. Archives
August 2023
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